Artificial tissue systems and uses thereof

ABSTRACT

An implant system and a method for controlling the natural and artificial microenvironments surrounding an implanted device using an artificial tissue system (ATS) and includes methods of diagnostic and testing related thereto. The ATS, among other things, induce better integration, function, and extended lifespan of the devices at the site of implantation. The ATS includes cells, such as naturally occurring, engineered, and/or artificial cells; matrices such as natural, engineered, artificial and/or hybrid matrices; tissue response modifiers (TRM); and/or cell response modifiers (CRM). The specific composition of the ATS is based on the nature of the tissue in which ATS-device combination is implanted and the nature of the implant device, as well as the required function and lifespan of the implanted device. Additionally, the ATS, as well as ATS-device combinations can be utilized in vitro to aid in the design of improved ATS, devices and ATS-device combinations for in vivo uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.10/578,171 filed May 4, 2006, which is the Section 371 U.S. NationalStage of PCT/US04/37302 filed Nov. 5, 2004, which claims the benefit ofU.S. Provisional Application No. 60/518,412, filed Nov. 7, 2003, thecontent of which is incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application incorporates by reference the sequence listing havingthe file name mtt102us_sequence_ST25 created Jun. 24, 2013, with a filesize of 2kb.

STATEMENT WITH REGARD TO FEDERAL SPONSORED RESEARCH & DEVELOPMENT

This invention was made with Government support under Grants awarded bythe National Institutes of Health (1RO1RR14171) the United StatesDepartment of Defense W81XWH-04-1-0002 and W81XWH-04-1-0313. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the field of medical implants. Inparticular, this invention relates to apparatus and methods for bettercontrolling the natural and artificial microenvironments surroundingimplanted devices and sensors in biological systems.

BACKGROUND OF THE INVENTION

Implantable materials and devices, such as drug delivery systems,pacemakers, artificial joints, and organs play an important role inhealth care today. In addition to these devices, implantable monitoringdevices implantable sensors have great potential for improving both thequality of care and quality of life of patients and animals. Potentiallythese sensors can measure a wide variety of analytes in the blood andtissue, which would be critical in the early diagnosis and treatment ofdisease.

Unfortunately the development of these implantable sensors has beenhampered by the inability of currently designed implantable sensors toovercome their loss of function and short lifespan in vivo. Frequentlythis loss of function is a result of the acute and chronic tissuereactions to the implanted sensors. These tissue reactions are a resultof various factors including 1) tissue injury and inflammation as aresult of tissue trauma from the surgical implantation of the device, 2)immune and non-immune inflammation at the implantation site as a resultof “foreign body” reactions to the device, 3) the release to tissue oftoxic factors from the function of the device and or the chemicalbreakdown of the device and its coating. Ultimately, these chronicinflammatory reactions at sites of sensor implantation result in tissuedestruction and fibrosis and complete loss of sensor function in vivo.

Conventionally, efforts to extend the in vivo lifespan of implantableglucose sensors have focused on the uses of various sensor coatings, inan effort to “hide or stealth” the sensor from detection and theresulting tissue reactions. Unfortunately these approaches have not beensuccessful, and the use of various coatings has seen limited successbecause of the body innate and acquired host defense systems (immunity)that can detect minute differences between normal tissue elements andforeign materials such as sensor coatings.

Alternative conventional efforts to incorporate bioactive drugs andpeptides and proteins into the coatings or sensor associated drugdelivery systems have seen some success. However, in the case of sensorcoatings it has been found that 1) frequently only “analyte permeablecoatings” can be used as sensor coatings, thus limiting the type ofcoating available for implantable sensors; 2) binding of sufficientquantities of bioactive agents such as peptides and proteins, can bedifficult and often they do not remain active after being bound to thesensor coating; 3) the intense tissue reactions (proteins and cells)frequently “mask” or degrade the bioactive agents on the coatings andlimit their effectiveness 4) because of the limited quantities ofbioactive agents that can be incorporated into sensor coatings, thecoating and therefore the sensor have a limited lifespan in vivo andmust be replaced frequently. Additionally the device and its byproductscan also damage both the tissue and the sensor and its coatings. Forexample, implantable sensors generally function based on the use ofglucose oxidase which is specific for glucose. The enzyme needs to beimmobilized on the platinum wire by using a carrier protein such asalbumin and toxic crosslinking agent such as glutaraldehyde.Additionally, the glucose oxidase used in the sensor continuously breaksdown glucose into gluconic acid and hydrogen peroxide, both of which aretissue toxic as well as potentially “sensor toxic”. The hydrogenperoxide is further broken down in reactive oxygen radicals, which arealso toxic.

In the case of traditional drug delivery systems such as micro beads,they frequently do not incorporate (load) and or release bioactiveagents in quantities and for durations that are useful for implantablesensors. Since the drug delivery system used with implantable sensorsare usually located near the sensor, “foreign body” tissue reactions tothe drug delivery system often have negative “bystander” effects on thesensor and its function. For example, the breakdown of the drug deliverysystems such as micro beads result in the release of tissue toxic andsensor toxic byproducts that hinder sensor function in vivo. Ultimatelythe combination of inflammation, fibrosis and loss of blood vessels alsodecrease tissue levels of both glucose and oxygen, both essential toglucose sensor function in vivo, with a resulting loss of sensorfunction and lifespan in vivo. Clearly in the future, new approaches,methods and devices are needed to extend the function and lifespan ofimplantable sensors such as the glucose sensors used in conjunctionwith, for example, the disease condition known as diabetes.

Diabetes is a chronic disease that afflicts over 18 million people inUnited States, with an annual cost of $132 billion in direct andindirect expenditures in the United States. Diabetes is a leadingcontributor to many other diseases, including heart disease, stroke,blindness, kidney failure, and peripheral neuropathy. The key factor inpreventing these devastating complications of diabetes is the closemonitoring of blood glucose levels. Currently, repeated “finger sticks”to obtain capillary blood samples is the major approach to monitoringblood glucose levels. Unfortunately, because of the pain andinconvenience of this procedure associated with “finger sticking” thepatient compliance is often poor. However, even with good patientcompliance with regular blood glucose testing, it appears that bloodglucose swings often stay undetected. For example, initial continuousglucose monitoring has shown that glucose concentrations are only withina target range of 4-10 mmol/l for about 35 percent of the time. Clearly,there is a critical need for a method that would allow continuous bloodglucose monitoring in vivo.

Although implantable glucose sensors have been in existence for over 30years, and in vitro studies have demonstrated that they can function forweeks to months, glucose sensor function in vivo has seen littlesuccess. Previous in vivo studies have indicated that implantableglucose sensors lose function within hours to days after implantation.

It is generally accepted that the loss of sensor function in vivo isassociated with sensor induced tissue injury, inflammation and fibrosiswith associated blood vessel regression. Presently there has been littleprogress in the developments for the reason described above. In factcurrently available glucose sensors display rapid loss in sensorfunction within 1-3 days post sensor implantation, and even thesesensors require frequent reference “finger sticks” to determine bloodglucose levels to utilize to recalibrate the implanted sensor. Clearly anew approach and devices are needed for enhancing the in vivo functionand lifespan of implanted sensors such as the glucose sensor.

SUMMARY OF THE INVENTION

Briefly stated, the present invention in a preferred form includes anapparatus and a method for controlling the natural and artificialmicroenvironments surrounding an implanted device using an artificialtissue system (ATS). The ATS, among other things, induce betterintegration, improved function, and an extended lifespan of the devicesat the site of implantation. The ATS includes cells, such as naturallyoccurring, engineered, and/or artificial cells and matrices such as anatural, engineered, artificial and/or hybrid matrix. Tissue responsemodifiers (TRM) and/or cell response modifiers (CRM) as alsopreferentially included. The specific composition of the ATS is based onthe nature of the tissue in which also are preferred included theATS-device combination is implanted and the nature of the implanteddevice, as well as the required function and lifespan of the implanteddevice. Additionally, the ATS, as well as ATS-device combinations can beutilized in vitro to aid in the design of improved artificial tissuesystems, devices and ATS-device combinations for in vivo uses.

The invention includes an artificial tissue system, comprising (a) amatrix configured for biological contact with an implantable device, and(b) a plurality of cells supported by said matrix, said cells promotinga biological interaction between said implantable device and abiological system.

The invention also includes an implant system comprising an implantabledevice, a matrix in biological contact with said implantable device, anda plurality of cells supported by said matrix, said cells promoting abiological interaction between said implantable device and a biologicalsystem.

The invention includes an artificial implant system in biologicalcontact with a biological system comprising a cellular component, saidcellular component includes at least one cellular community whichinduces a biological response in the biological system. Also included isa matrix material, said matrix material being associated with a portionof the cellular community; and an implant device having a biologicalinterface wherein said biological interface is associated with thematrix material and the biological system.

The invention further includes a method of implanting a device in abiological system, comprising the steps of: obtaining said device,obtaining a matrix, placing said device in biological contact with saidmatrix, inserting cells into said matrix, said cells being capable ofpromoting a biological interaction between said implantable device andsaid biological system, and implanting said matrix into said biologicalsystem.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will be evident to one ofordinary skill in the art from the following detailed description withreference to the accompanying drawings, in which:

FIG. 1 shows an artificial tissue system with various possibleconstituents consistent with the present invention;

FIGS. 2A-2E show the morphology of various control and Rous Sarcomavirus infected DF-1 cells consistent with the present invention;

FIGS. 3A and 3B respectively show the in vitro expression of p27 andVEGF in both control and RCAS infected DF-1 cells consistent with thepresent invention;

FIG. 4 shows GFP:DF-1 cells associated with a fibrin matrix materialconsistent with the present invention;

FIGS. 5A and 5B respectively show in vitro VEGF protein expression andAvian Leucosis p27 expression associated with cells in a fibrin matrixmaterial at various fibrinogen concentrations consistent with thepresent invention;

FIGS. 6A and 6B show the impact of thrombin on VEGF:DF-1 cells andcontrol cells consistent with the present invention;

FIGS. 7A-7E respectively show the development of the chorioallantoicmembrane in the ex ova model of the chick embryo at days 3, 7, 10, 14,and 17;

FIGS. 8A-8C respectively show the histology of portions of normalchorioallantoic membrane at days 8, 12, and 16;

FIGS. 9A-9D respectively show gross views of the inflammatory responsesin an ex ova chorioallantoic membrane model of LPS/india ink inducedreactions at days 0, 1, 4, and 8 consistent with the present invention;

FIGS. 9E-9H respectively show histologic views of the inflammatoryresponses in an ex ova chorioallantoic membrane model of LPS/india inkinduced reactions at days 0, 1, 4, and 8 consistent with the presentinvention;

FIGS. 10A-10D respectively show gross views of the inflammatoryresponses in an ex ova chorioallantoic membrane model of cotton threadinduced reactions at days 0, 1, 4, and 8 consistent with the presentinvention;

FIGS. 10E-10H respectively show histologic views of the inflammatoryresponses in an ex ova chorioallantoic membrane model of cotton threadinduced reactions at days 0, 1, 4, and 8 consistent with the presentinvention;

FIGS. 11A-11D respectively show gross views of the tissue response in achorioallantoic membrane associated with nylon at days 0, 1, 4, and 8consistent with the present invention;

FIGS. 11E-11H respectively show histologic views of the tissue responsein a chorioallantoic membrane associated with nylon at days 0, 1, 4, and8 consistent with the present invention;

FIGS. 12A-12D respectively show gross views of the tissue response in achorioallantoic membrane associated with silastic tubing at days 0, 1,4, and 8 consistent with the present invention;

FIGS. 12E-12H respectively show histologic views of the tissue responsein a chorioallantoic membrane associated with silastic tubing at days 0,1, 4, and 8 consistent with the present invention;

FIGS. 13A and 13B show the gross appearance of GFP:DF-1 cells grown onnylon disks and placed on a chorioallantoic membrane consistent with thepresent invention;

FIGS. 14A-14D respectively show the gross views, under bright fieldmicroscopy, of GFP:DF-1 cells grown on nylon disks or placed in nylonrings on a chorioallantoic membrane at days 4, 6, and 8 consistent withthe present invention;

FIGS. 14E-14H respectively show the gross views, under fluorescentmicroscopy, of GFP:DF-1 cells grown on nylon disks or placed in nylonrings on a chorioallantoic membrane at days 4, 6, and 8 consistent withthe present invention;

FIGS. 14I-14L respectively show the histologic views of GFP: DF-1 cellsgrown on nylon disks or placed in nylon rings on a chorioallantoicmembrane at days 4, 6, and 8 consistent with the present invention;

FIGS. 15A-15D respectively show gross views of neovascularizationinduced in the ex ova chorioallantoic membrane model using VEGF:DF-1cells grown on nylon disks consistent with the present invention;

FIGS. 15E-15H respectively show histologic views of neovascularizationinduced in an ex ova chorioallantoic membrane model using VEGF:DF-1cells grown on nylon disks consistent with the present invention;

FIGS. 16A and 16B respectively show gross views of neovascularizationinduced in an ex ova chorioallantoic membrane model using AS-VEGF:DF-1and VEGF:DF-1 cells added to nylon rings consistent with the presentinvention;

FIGS. 16C and 16D respectively show histologic views ofneovascularization induced in an ex ova chorioallantoic membrane modelusing AS-VEGF:DF-1 and VEGF:DF-1 cells added to nylon rings consistentwith the present invention;

FIGS. 17A-17D show the gross morphologic appearance ofneovascularization induced in the ex ova chorioallantoic membrane modelusing VEGF:DF-1 cells in a fibrin matrix material consistent with thepresent invention;

FIGS. 18A and 18B, respectively, are a top view of a sensor and a sideview of a sensor consistent with the present invention;

FIG. 18C is graph showing the relationship between a sensor output andchemical concentration consistent with the present invention;

FIG. 18D shows a sensor as shown in FIGS. 18A and 18B associated with anin vitro experimental set-up consistent with the present invention;

FIGS. 19A and 19C respectively show a chemical sensor incorporated in anex ova chorioallantoic membrane model and the associated sensor responsegraph consistent with the present invention;

FIGS. 19B and 19D respectively show a chemical sensor incorporated in anex ova chorioallantoic membrane model and the associated sensor responsegraph consistent with the present invention;

FIG. 20 is a graph quantifying an acetaminophen sensor response in an exova chorioallantoic membrane model consistent with the presentinvention;

FIG. 21 is graph of the impact of mVEGF:DF-1 cell inducedneovascularization on an acetaminophen sensor response in an ex ovachorioallantoic membrane model consistent with the present invention;

FIG. 22 is SEQ. ID NO 1 and shows a nucleic acid sequence associatedwith mouse vascular endothelial growth factor M95200;

FIG. 23 is a needle type acetaminophen sensor consistent with thepresent invention;

FIG. 24 is a graph of a needle type acetaminophen sensor consistent withthe present invention;

FIG. 25 is a photomicrograph of a glucose sensor with a referenceelectrode and a working electrode consistent with the present invention;

FIG. 26 is a graph of a glucose sensor response in vivo consistent withthe present invention;

FIGS. 27A and 27D show an implanted glucose sensor in an ex ova modelconsistent with the present invention;

FIGS. 27B and 27C show glucose sensor response associated with an ATSwith GFP:DF-1 consistent with the present invention;

FIGS. 27E and 27F show glucose sensor response associated with an ATSwith VEGF:DF-1 consistent with the present invention;

FIG. 28 is a comparative graph of glucose sensor responses using ATS inan ex ova CAM model under various treatments consistent with the presentinvention.

FIGS. 29A-29H show the histopathologic views of tissue reactions inducedin ex ova CAM tissue by ATS with GFP:DF-1 and VEGF:DF-1 cells consistentwith the present invention;

FIG. 30 is a graph of a glucose sensor response for a sensor, which isassociated with an ATS having genetically engineered cells consistentwith the present invention;

FIGS. 31A-31C show the implantation of a glucose sensor in a mouse.

FIG. 32 show various graphs associated with the simultaneous monitoringof a subcutaneous sensor and blood glucose over a 30 day periodconsistent with the present invention;

FIG. 33 is a graph showing the mean loss of sensitivity for a sensor;

FIG. 34 is a graph showing sensor functionality loss in vivo;

FIGS. 35A, C, E, and G respectively show histologic views of IRC mousetissue, stained with HE, associated with a working electrode of animplanted glucose sensor at 1 dpi, 3 dpi, 7 dpi, and 14 dpi consistentwith the present invention;

FIGS. 35B, D, F, and H respectively show histologic views of IRC mousetissue, stained with trichrome, associated with a working electrode ofan implanted glucose sensor at 1 dpi, 3 dpi, 7 dpi, and 14 dpiconsistent with the present invention;

FIGS. 35I, K, M, and O respectively show histologic views of IRC mousetissue, stained with HE, associated with a reference electrode of animplanted glucose sensor at 1 dpi, 3 dpi, 7 dpi, and 14 dpi consistentwith the present invention;

FIGS. 35J, L, N, and P respectively show histologic views of IRC mousetissue, stained with trichrome, associated with a reference electrode ofan implanted glucose sensor at 1 dpi, 3 dpi, 7 dpi, and 14 dpiconsistent with the present invention;

FIG. 36 shows a histologic view of mouse model tissue, which includesthe presence of giant cells in association with a glucose sensor at 7dpi consistent with the present invention;

FIGS. 37A-37H show histologic views of tissue at 30 dpi associated withimplanted sensors having a working electrode, an intermediate wire, anda reference electrode consistent with the present invention;

FIG. 38 is a graph showing function of glucose sensor in a mouse modelconsistent with the present invention;

FIG. 39 show various graphs associated with blood glucose and sensorresponse in both control and dexamethasone treated mice at time points 1hr, 4 hr, and 8 hrs consistent with the present invention; and

FIG. 40 is a diagram showing utilization of endothelial stem cellsconsistent with the present invention.

FIGS. 41A-C are histologic views of neovascularization induced inimmunodeficient mice (nude/nude) by subcutaneous injection of VEGF:DF-1containing ATS consistent with the present invention.

FIG. 42 shows a human coxsackievirus and adenovirus receptor (hCAR)consistent with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The general meaning of the following terms as used in the presentapplication, unless specifically modified, are: “Normal Cells”:biological cells derived from living organisms, and/or tissues, whichretain a normal genotype and phenotype, usually obtained directly fromtissue or from primary culture. “Mutant Cells”: biological cells withspontaneously altered genotype and phenotype, such as cancer cells, cellderived from naturally occurring genetically deficient organisms,usually obtained in secondary culture and or continuous cell lines.“Engineered Cells”: genetically or chemically modified biological cells(usual original source is Normal or Mutant cells). “Transgenic Cells”:biological cells derived from transgenic animals, in which the cellshave genetically induced alterations of genotype and or phenotype. “GeneTransfer Cells”: biological cells that have altered phenotype resultingin alteration of cell structure and or function. This includesknockouts, knockdowns, “over-expressors” etc. “Chemically ModifiedCells”: biological cells in which membrane, cytoplasm structural ornucleolar elements of the cell are altered permanently or for extendedperiods, thus altering cell structure and or function. “ArtificialCells”: biological cells lacking the ability to replicate but capable ofsensing and responding to their microenvironment. For example enucleatedcells, or cells lacking a nucleus (e.g. red blood cells), in whichgenetic elements such as DNA, RNA, viral vectors, nanodevices ornanomaterials can be incorporated for in vivo uses. Hybrid Cells:biological cells that are the result of cells fusion, and orcombinations of engineered and or artificial cells. “Matrix material”:complex heterogeneous networks of insoluble macromolecules such asglycoproteins, carbohydrates, structural proteins (e.g. collagen), aswell as bound proteins and factors. These matrices contain specificbinding sites for cells, factors (e.g. cytokines and growth factors) andproteins, which directly control cell adhesion and function in vivo andin vitro. “Biological Matrices”: matrices obtained from organisms,tissues, or cell. Examples of biological matrices include interstitialmatrices, basement membrane, fibrin clots. Interstitial matrices aregenerally composed of fibrillar and nonfibrillar collagen, elastin,fibronectin proteoglycans, hyuronate, as well as other components.Basement membranes are composed of nonfibrillar collagen (usually IV),laminin, heparin sulfate, proteoglycan, and other glycoproteins. Fibrinclots are complex networks of plasma proteins including fibrin(ogen),fibronectin, glycoproteins, heparin, thrombin collagen, as well as otherplasma proteins cross-linked to the fibrin clots via Factor XIII.Additionally, fibrin clots have extensive binding sites for variousfactors and cells including leukocytes, fibroblasts and endothelialcells. “Engineered Matrices”: genetically and or chemically modifiedbiological matrices. “Hybrid matrices”: combinations of biological,engineered and or artificial matrices. In addition, the meaning ofvarious abbreviations as used within the present application, unlessspecifically modified, include ES, embryonic stem cell; MSC, mesenchymalstem cell; MAPC, multipotent adult progenitor cell; HSC, hematopoieticstem cell; NSC, neural stem cell; NPC, neural progenitor cell; MDSC,muscle-derived stem cell; ECM, extracellular matrix; EGF, epidermalgrowth factor; LIF, leukemia inhibitory factor; SCF, stem cell factor;HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor;VEGF, vascular endothelial growth factor; BMP, bone morphogeneticprotein; BDNF, brain-derived neurotrophic factor; NT, neurotrophin;CNTF, ciliary neurotrophic factor; bFGF, basic fibroblast growth factor;TGF-β, transforming growth factor-beta; IL, interleukin; G-CSF,granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophagecolony stimulating factor; IGF, insulin-like growth factor; RA, retinoicacid; and FBS, fetal bovine serum.

An artificial tissue system implant, in one embodiment of the invention,as shown in FIG. 1, includes an implant device, for example, a sensor10. The sensor 10 is surrounded by an artificial tissue system 12. Theartificial tissue system (ATS) 12 includes cells. For example, the cellmay be biological cells 14, genetically engineered cells 14 a,artificial cells 14 b, stem cells 14 c and/or support cells 14 d. Thesupport cells 14 d generally are included with other cells and serve toprovide nutrients, factors, physical surfaces, or other required ordesirable products to the cells they support. The ATS may also includegenetic elements 16, cell response modifiers (CRM) 18, and/or tissueresponse modifiers (TRM) 20. The ATS 12 further includes a matrixmaterial 22. The matrix material 22 may be a natural and/or syntheticmaterial. For example, the matrix material 22 may include biologicalmatrices such as naturally occurring matrices that occur in viableorganisms (in vivo) and tissues including ex vivo tissues, as well as inassociation with cells maintained in vitro, or combinations thereof. Onecharacteristic of the matrix material 22 is the ability to provide athree dimensional structure to the ATS 12. This three dimensionalstructure provides a volume of space that allows for biological contactwherein various components of the ATS 12, sensor 10, and surroundingtissues can biologically associate with one another. For example, thematrix material may provide the necessary framework in which variouscells can be secured as well as providing for the movement of nutrients,chemicals, and other bioactive agents to, from, and/or between cells,tissues, and/or an implant device, such as, the sensor 10. In addition,the matrix material 22 is in biological contact with portions of theimplant device and the surrounding biological system, if present. Itshould be understood that the biological contact includes, among otherthings, chemical, liquid, gas, and/or mechanical contact. For example,cellular tissue of the biological system may intrude, or otherwiseextend physically into, the volume of space occupied by the matrixmaterial. This cellular tissue may also be in physical, chemical, and/orfluid contact with the cells, portions of the implant device, such asthe sensor 10, genetic elements 16, CRM 18, and/or TRM 20. The geneticelements 16 include, for example, agent(s) that directly cause thetemporary or permanent change of the genetic composition or expressionof a cell or tissue, or indirectly cause the temporary or permanentchange of the genetic composition or expression of a cell or tissue. Forexample, single or double strand DNA, single or double strand RNA,plasmids, viral vectors, and/or DNA or RNA viral vectors. In addition,it should be understood that the ATS may be formed into, for example,any biologically relevant shape, for example, a tube, sponge, sphere,strand, coiled strand, capillary network, film, fiber, mesh, and/orsheet.

In one embodiment of the invention, the cells include eukaryotic cells;prokaryotic cells; vertebrates cells; invertebrates cells; normal cells;cancer cells; mutant cells; engineered cells, such as geneticallyaltered cells, chemically altered cells, transgenic cells, hybrid cells(hybridomas); artificial cells; and stem cells, such as embryonic stemcells, adult stem cells, stem cell lines, engineered stem cells. Thecells may be classified as categories of functional cells, for example,inflammatory cells, immune cells, tissue cells, cells which controlwound healing, cells which control fibrosis, cells which control tissueregeneration, regulatory cells, cytokine producing cells, growth factorproducing cells, matrix producing cells, vascular cells, connectivetissue cells, bone producing cells and bone, blood cells. The cells mayalso be classified as types of cells, for example, endothelial cells,fibroblasts, epithelial cells, muscle cells, fat cells, lymphocytes,macrophages, mast cells, polymorphonuclear leukocytes, red blood cells,neurologic cells, osteoblasts, osteoclasts, nerve cells, fat cells,brain cells. Other categories of cells may be used and include, but arenot limited to, autologous cells, heterologous cells, allogenic cells,xenogenic cells, autologous cells, (relative to the host), heterologouscells (relative to the host), allogenic cells (relative to the hosttissue), xenogenic cells (relative to the host tissue). It should beunderstood that the cells may be used in combination with one anothersuch that a cellular component is formed. The cellular component mayinclude one or more cellular communities wherein the communitiesinteract on, for example, symbiotic, commensal, saprophytic, inhibitoryand/or other biologically relevant association. For example, engineeredand non-engineered cells may be used in combination to provideadvantageous biologic contact with one another and with a biologicsystem with which they are associated, for example a living mammalbiologic system.

In one embodiment of the invention, cell of different categories and/ortypes may be combined in the matrix material 22. For example, functionalcells may be used which regulate the function of other cells within thematrix material 22. This may include cells that produce cytokines andgrowth factors; cells that regulate the function of the cells within thehost tissue; cells that include matrix producing cells within the hosttissue; cells that produce cytokines and growth factors which controlcells in the host tissues; cells that controls inflammation within theATS; cells that control wound healing within the ATS; cells that controlfibrosis within the ATS; cells that control neovascularization withinthe ATS; cells that control cell proliferation within the ATS; cellsthat control immune responses within the ATS; cells that include cellsthat control cell death within the ATS; cells that includes cells thatcontrol inflammation within the tissues; cells that control woundhealing within the tissues; cells that control fibrosis within thetissues; cells that control neovascularization within the tissues; cellsthat control cell proliferation within the tissues; cells that controlimmune responses within the tissues; cells that control cell deathwithin the tissues; cells that produces cytokines; cells that producegrowth factors; cells that control vessel formation and regression;cells that produce genetically altered proteins and peptides; and cellsthat overproduce proteins and/or peptides.

Sources of biological cells include cells directly isolated from in vivosources; cells obtained from embryonic tissues, neonatal tissues,juvenile or adult tissues; cells obtained from in vitro sources; cellsobtained from primary cell culture sources; cells obtained fromsecondary cell culture sources; and cells obtained from continuous celllines.

In one embodiment of the invention the CRM 18 and/or TRM 20 aredifferentiated based on their biologic effect. For example, “cellresponse modifiers” (CRM) 18, as used herein, include agents thatcontrol the structure and or function of cells in vitro and or in vivo,whereas, “tissue response modifiers” (TRM) 20 as used herein, includeagents that control the structure and or function of tissues in vivo andor ex vivo. The CRM 18 may include cells genetically engineered andnon-genetically engineered: biological cells, synthetic cells,regulatory cells, tissue support cells, mutant cells, artificial cells,genetically altered cells, chemically altered cells, and/or stem cells.The CRM 18 may control cellular proliferation; cell injury; cell death;cell metabolism; cell protein synthesis; cell gene expression; and/oragents that control the structure and/or function of cells derived fromany in vitro or in vivo source.

In one embodiment of the invention, the categories or types of cellswhose structure and or function is controlled by CRM 18, include cellsderived from embryonic, neonatal, juvenile and or adult cells. Inaddition, cells that may be controlled by CRM 18 include biologicalcells, eukaryotic cells, prokaryotic cells, vertebrates' cells,invertebrates cells, normal cells, cancer cells, mutant cells,engineered cells, artificial cells, stem cells, and/or hybrid cells. Inaddition, cells controlled by CRM 18, include, for example, endothelialcells, fibroblasts, epithelial cells, muscle cells, fat cells,lymphocytes, macrophages, mast cells, polymorphonuclear leukocytes, redblood cells, neurologic cells, osteoblasts, osteoclasts, nerve, fatcells, brain cells, bone cells, tissue derived stem cells, blood derivedstem cells, bone derived stem cells.

In one embodiment of the invention the CRM 18, include agents that, forexample, control cell homeostasis by controlling cell functions such ascell activation, cell proliferation, cell metabolism, cell death(including apoptosis), cell differentiation and maturation, cell size,cell composition.

In one embodiment of the invention, the TRM 20 includes, for example,agent(s) that control tissue growth; tissue differentiation; tissueinjury; innate immune responses; acquired immune responses; humoralimmune responses; cell mediated immune responses; inflammation; acuteinflammation; chronic inflammation; wound healing; regeneration; tissuerepair; neovascularization; bone destruction; bone injury, repair and orregeneration; connective tissue destructions; controls connective tissueinjury, repair and regeneration; fat tissue injury, repair and orregeneration; neurologic tissue injury, repair and or regeneration;and/or responses using TRM 20. The TRM 20 may include: cell to cellprotein transporter molecules; antibodies; proteins, modified proteinsand/or recombinant protein; chemicals; drugs; genetic elements;recombinant DNA; RNAs, including siRNA; altered RNAs; geneticallyaltered RNAs; chemically altered RNAs; DNA; altered DNAs; carbohydrates;lipids and fatty acids; radiation energy; magnetic energy; viruses;single or double strained DNA; and/or single or double strained RNA.

The TRM 20 may be used in combination, for example, the TRM 20 mayinclude: TRM that controls tissue injury and a second TRM that controlsinflammation; TRM that controls inflammation and a second TRM thatcontrols fibrosis; TRM that controls inflammation and a second TRM thatcontrols neovascularization; TRM that controls inflammation and a secondTRM that controls tissue regeneration; TRM that controls cell injury anda second TRM that controls inflammation; TRM that controls cell deathand a second TRM that controls inflammation; TRM that controlsinflammation and a second TRM that controls fibrosis; TRM that controlsinflammation and a second TRM that controls neovascularization; TRM thatcontrols fibrosis and a second TRM that controls neovascularization;and/or TRM that controls inflammation and a second TRM that controlstissue regeneration.

The TRM 20 may, for example, in one embodiment of the invention includethe agents 2-(3-benzophenyl)propionic acid,9-alpha-fluoro-16-alpha-methylprednisolone, methyl prednisone,fluoroxyprednisolone, 17-hydroxycorticosterone, cyclosporin,(+)-6-methoxy-.alpha.-methyl-2-naphthalene acetic acid,4-isobutyl-.alpha.-methylphenyl acetic acid, Mitomicyin C,Acetaminophen, Dexamethasone, Diphenyhdramine, Hydrochloride, Cromolyn,3-(1H-Tetrazol-5-yl)-9H-thiol-xanthene-9-one 10,10-dioxide monohydrate,H1 and H2 histamine antagonists (H1 antagonists: mepytramine ortriprolidine) transforming growth factor alpha, anti-transforming growthfactor beta, epidermal growth factor, vascular endothelial growthfactor, anti-transforming growth factor beta antibody, anti-fibroblastantibody, anti-transforming growth factor beta receptor antibody,arginine-glycine-aspartic acid, REDV, or a combination thereof.

Categories of tissues whose normal structure and or function iscontrolled by TRM, include, for example, biological tissues ofvertebrates, invertebrates; normal tissue; injured tissue; regeneratingtissue; repairing tissue; cancer tissue; mutant tissue; engineeredtissue; artificial tissue; stem cell tissues; hybrid tissues;endothelial tissue; fibroblasts; epithelial tissue; muscle tissue; fattissue; lymphocytes; macrophages; mast tissue; polymorphonuclearleukocytes; red blood cells, soft tissue; neurologic tissue;osteoblasts; osteoclasts; nerve; brain tissue; bone tissue; tissuederived stem tissue; blood derived stem tissue; and/or bone derived stemtissue.

Categories of tissues whose structure or function is controlled by TRMex vivo include, for example, tissues originally derived from embryonic,neonatal, juvenile and/or adult tissues. Categories of tissues whosestructure or function is controlled by TRM in vivo and or ex vivoinclude, for example, embryonic tissues, neonatal tissues, juvenile oradult skin. Injured tissues controlled in vivo and or ex vivo by TRM,include, for example, normal embryonic tissues, neonatal tissues,juvenile or adult skin. Tissues controlled in vivo and or ex vivo byTRM, include, for example, include embryonic tissues, neonatal tissues,juvenile or adult soft tissue, hard tissue, e.g. bone), skin, cardiacsystem, pulmonary, hepatic, gastrointestinal tract, biliary tract,urinary tract, genital tract, vision, neurologic or endocrine systems,blood vessels, bones, joints, tendons, nerves, muscles, the head, theneck, or any organ system or combinations there of.

In one embodiment of the present invention factors that are used tocontrol vascular endothelial cell function in vitro (i.e. cell responsemodifiers 18) also may induce or suppress new blood vessel formation invivo thus under the right circumstances they are also tissue responsemodifiers 20. For example, these factors may include: VascularEndothelial Growth Factor (VEGF); Fibroblast Growth Factor (FGF);Interleukin-8 (IL-8); Angiogenin; Angiotropin; Epidermal Growth Factor(EGF); Platelet Derived Endothelial Cell Growth Factor; TransformingGrowth Factor α (TGF-α); Transforming Growth Factor β (TGF-β); NitricOxide; Thrombospondin; Angiostatin; and Endostatin.

In one embodiment of the present invention, cell response modifiers 18are used, but because they also operates to control inflammation andimmune responses as well as development in vivo they are also examplesof cell response modifiers that can act in vivo as tissue responsemodifiers 20. For example, cytokines and growth factors included in thisoperative definition include: TH1/TH2 Interleukins (IL-2, IL-4, IL-7,IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17); the IL-1 family(IL-1-alpha, IL-1Ra, IL-18, IL-1-beta); the TNF family, for example TNFLigand and TNF/NGF Receptor Families, TNF alpha, Lymphotoxin alpha andbeta, Fas Ligand, CD40 Ligand, CD30 Ligand, CD27 Ligand, RANK LigandApo2L/TRAIL; the IL-6 family, for example, IL-6 Ligand and ReceptorFamily, IL-6, 1L-11, Oncostatin M, CT-1; macrophage activation, such as,IFNalpha, IFN beta, and IFNomega Ligands, IFNgamma, Osteopontin, MIF;TGF beta, BMP Family, PDGF, VEGF, Poxvirus Vascular Endothelial GrowthFactor (VEGF) Homologs of Orf Virus, Angiostatin, Activin, Endostatin,Methoxyestradiol, Poxvirus Growth Factors Related to EGF; IL-3, IL-5,Stem Cell Factor, GM-CSF CSF-1, G-CSF, Erythropoietin, Thrombopoietin;MGSA/GRO, ENA-78, IL-8, H. GCP-2, A. CTAP-III, betaTG, and NAP-2,Platelet Factor 4, IP-10 MIG, SDF-1, BLR1 Ligand/BCA-1/BLC, 9E3/cCAF;MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5 RANTES, 1-309, MIP-alpha,MIP-beta, Eotaxin, PARC, Eotaxin 2, MIP-gamma/MRP-2, Mu C10, Leukotactin1, CKbeta8-, B. HCC-1, SLC (6CKine), ELC, H TECK/CCL25, CC Chemokine ofMolluscum Contagiosum Virus, Lymphotactin, Fractalkine, PoxvirusSecreted Complement Control Proteins; IL-2 Family Receptors, IL-2Receptor, IL-4 Receptor, IL-7 Receptor, IL-9 Receptor, IL-10 Receptor,IL-12 Receptor, IL-13 Receptor, IL-15 Receptor, IL-16 Receptor (CD4),IL-17 Receptor, Prolactin Receptor; IL-1 Family Receptors, such as, IL-1Receptor Family, IL-1 Receptor Type I, Poxvirus IL-1beta ReceptorHomologs, IL-18 Receptor, IL-1 Receptor Type II; TNF Receptors, PoxvirusTNF Receptor Homologs, Lymphotoxin beta Receptor, Fas, CD40, CD30,4-1BB, RANK, Osteoprotegerin, CD27, HVEM, DR4, DRS, DcR1, DcR2, DcR3,Ox40, GIT Receptor; IL-6 Receptor; IL-11 Receptor, OSM Receptor, CT-1Receptor; IFNgamma Receptor, Poxvirus IFNgamma Receptor Homologs, IFN cbeta Receptor, Poxvirus IFN c beta Receptor Homologs, OsteopontinReceptor, TGF beta Receptors, BMP Receptor, Hematopoietic Receptors, forexample the Hematopoietic Receptor Family of IL-3 Receptor, IL-5Receptor, SCF Receptor, GM-CSF Receptor, G-CSF Receptor, TPO Receptor;CXC Chemokine Receptors, such as, CXCR1 and CXCR2, CXCR3, CXCR4, CXCR5,R. CC, C, and CX3C; CC Chemokine Receptors, such as, CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, CCR8, D6, ECRF3, Poxvirus Membrane-bound GProtein-coupled Receptor Homologs, US28, Kaposi's Sarcoma-associatedHerpesvirus GPCR, DARC, CX3CR1, Poxvirus Secreted Chemokine-bindingProteins, CCR9, XCR1; and Miscellaneous non-Cytokine ProinflammatoryFactor Receptor, such as C5a Receptor, C3a Receptor, PAF Receptors, fMLPReceptors, Opioid mu, delta, and kappaReceptors for Endorphins, LipoxinA4 Receptor, ACTH Receptor, BLTR: the Leukotriene B4 Receptor, PACAP andVIP Receptors, Lysophospholipid Growth Factor Receptors.

In one embodiment of the present invention the matrix material 22 mayinclude: basement membranes, for example Matrigel™; fibrin clots,including plasma derived clots; collagens, for example, fibrillarcollagens (types I, II, III, V and IX collagen); basement membranecollagen, such as type IV collagen; other collagens (types VI, VII, IX,XVII, XV and XVIII collagen); fibronectin; laminin; proteoglycans;glycoproteins; glycoaminoglycans; elastins; hyaluronan; adhesiveglycoproteins; mucins; and polysaccharides. In some cases, certainfactors can be included with the matrix material 22 to advantageouslyenhance the characteristics of the matrix material 22 and/or itsproduction. For example, factors that can be included are: TGF-beta;FGF; angiotensin II; Insulin-like growth factor; and Ascorbic acid.

In one embodiment of the present invention, the matrix material 22 isprimarily composed of fibrin. The matrix material 22 may also becomposed of a solubilized basement membrane preparation such asMatrigel™ as supplied from BD Biosciences. The solubilized basementmembrane, like fibrin, is a naturally occurring proteinmatrix/bio-hydrogel that has a wide variety of binding sites for cellsand factors. These factors may include growth factors and cytokines. Forexample, the solubilized basement membrane may include laminin,collagen, including collagen IV, heparin sulphate proteoglycans, andentactin. Solubilized basement membrane has been used extensively as acell matrix/depot in a wide variety of in vitro and in vivo studiesparticularly in the area of tumor cell biology and angiogenesis.

In one embodiment of the invention the solubilized basement membrane isa liquid at 4° C. but becomes a solid bio-hydrogel when warmed to 37° C.This ability to convert solubilized basement membrane from a liquid to asolid by simply raising the temperature, allows for a wide variety ofstrategies for entrapping genetically engineered cells, factors,proteins and genes. It should be understood that the terms entrap,entraps, entrapped, entrapping, and the like are intended to include forthe purpose of this application the concept that the matrix material 22provides a mechanical association with the biological cells and/or thatthe matrix material 22 provides specific binding sites for thebiological cells. For example, specific binding sites which includereceptor and/or adhesion sites.

In one embodiment of the present invention stem or progenitor cells 14 care included in the ATS. These stem or progenitor cells may be includedin a matrix material 22, which is selected based on the origin of thestem or progenitor cells. For example, expansion of undifferentiatedstem cells, in vitro, may accomplished with a gelatin matrix material;expansion of nestin+neural progenitor cells may be accomplished withlaminin, RA, Survival of embryonic stem cell derived motor neurons withbasement membrane, and endothelial cells with collagen IV. If, forexample, the stem or progenitor cells are of a bone marrow origin of theMSC, MAPC, or HSC type, then fibronectin and or basement membranes maybe used. For example, expansion in vitro of undifferentiated MAPCs withfibronectin; osteoblasts with fibronectin; endothelial cells withfibronectin; and hepatocyte-like cells: basement membranes. If, forexample, the stem or progenitor cells are of an adult tissue origin ofthe hepatic oval cell, NSC/NPC, adipose stem cell, or MDSC type, thenfibronectin, laminin and/or collagen may be used. For example, expansionof undifferentiated oval cells with fibronectin; hepatocyte withfibronectin; pancreatic islet with fibronectin; neuron, glial cells withfibronectin, laminin; expansion of MDSCs with collagen, and osteoblastwith collagen.

In one embodiment of the present invention, several growth factors orcytokines may be used as, for example, CRM 18 to promote stem orprogenitor cell proliferation and differentiation in vitro. For example,if the stem or progenitor cells are embryonic stem cells, then expansionof undifferentiated ES cells can be accomplished with LIF; pancreaticendocrine progenitor with bFGF; pancreatic islet with bFGF; expansion ofNestin+ neural progenitors with bFGF; RASurvival of ES-derived motorneurons with BDNF, NT-3, CNTF, GDNF; glial progenitor cells with bFGF,PDGF-AA; adipocyte13RAChondrocyte with BMP-2, BMP-4; dendritic cells:GM-CSF, IL-3; and endothelial cells with VEGF. If the stem or progenitorcells are derived from bone marrow and are of the MSC, MAPC, or HSCtypes, then, for example, osteoblast may be utilized with BMP-2, bFGF;chondrocyte with TGF-β3; neuron, glial cells with EGF, BDNF; expansionof undifferentiated MAPCs with EGF, PDGF-BB; chondrocyte with TGF-β1;endothelial cells with VEGF; hepatocyte-like cells with FGF-4, HGF; andplatelets, red/white blood cells with IL-3, IL-6, G-CSF. If the stem orprogenitor cells are derived from adult tissues and are of the Hepaticoval cell, NSC/NPC, Adipose stem cell or MDSC types, then, for example,expansion of undifferentiated oval cells can be accomplished with SCF,Flt-3 ligand, IL-3, LIF; hepatocyte with HGF, EGF; pancreatic islet withSCF, Flt-3 ligand, IL-3; expansion of NPCs with bFGF, EGF, LIF; neuron,glial cells with bFGF, EGF, PDGF-AA, PDGF-AB, PDGF-BB, NT-4, CNTF;osteoblast with TGF-β1; expansion of MDSCs with IGF-1, EGF, SCF, FGF2;and osteoblast: BMP-2.

In one embodiment of the present invention stem or progenitor cells 14 care promoted utilizing other factors as, for example, TRM 20. Forexample, if the stem or progenitor cells are embryonic stem cells, thenpancreatic islet cells can be utilized with nicotinamide; expansion ofNestin+ neural progenitors can be accomplished with poly-ornithine;neurons with poly-ornithine, RA; Adipocytes with RA; and osteoblastswith RA, dexamethasone, ascorbate, β-glycerol phosphate. If the stem orprogenitor cells are derived from bone marrow and are of the MSC, MAPC,or HSC types, then, for example, osteoblasts with dexamethasone,ascorbate, β-glycerol phosphate; chondrocytes with dexamethasone;neuron, glial cells with RA; adipocytes with dexamethasone, insulin,indomethacin, 1-methyl-3-isobutylxanthine; expansion of undifferentiatedMAPCs with 2% FBS; osteoblasts with dexamethasone, ascorbate, β-glycerolphosphate; platelets, red/white blood cells with erythropoietin,thrombopoietin. If the stem or progenitor cells are derived from adulttissues and are of the Hepatic oval cell, NSC/NPC, Adipose stem cell orMDSC types, then, for example, pancreatic islet cells can be utilizedwith nicotinamide; osteoblasts with Dexamethasone, ascorbate, β-glycerolphosphate; chondrocytes with insulin, ascorbate; and adipocytes withdexamethasone, insulin, indomethacin, 1-methyl-3-isobutylxanthine.

One embodiment of the present invention includes the use of implantablesensors 10, which include, for example, chemical sensors and biosensorssuch as glucose sensors. However, other devices may be used in additionto the sensor 10, or may replace the sensor 10. For example: bioreactorsfor liver, kidney or other organ support systems; catheters; artificialarteries; artificial organs; tissue fragment-containing devices;cell-containing devices; ligament replacements; bone replacements;coronary pacemakers; lap-bands, monitors; artificial larynxes;prostheses; brain stimulators; bladder pacemakers; shunts; stents;tubes; defibrillators; cardioverters; heart valves; joint replacements;fixation devices; ocular implants; cochlear implants; breast implants;neurostimulators; bone growth stimulators; vascular grafts; musclestimulators; left ventricular assist devices; pressure sensors; vagusnerve stimulators; drug delivery systems; sutures and staples. Inaddition the devices may include implants. For example: prostheses, suchas joint replacements; artificial tendons and ligaments; dentalimplants; blood vessel prostheses; heart valves; cochlear replacements;intraocular lens; mammary prostheses; penile and testicular prostheses;tracheal, laryngeal, and esophageal replacement devices; artificialorgans such as heart, liver, pancreas, kidney, and parathyroid; repairmaterials and devices such as bone cements, bone defect repairs, boneplates for fracture fixation; heart valves; catheters; nerveregeneration channels; corneal bandages; skin repair templates;scaffolds for tissue repair and regeneration; and devices such aspacemakers, implantable drug delivery systems (e.g., for drugs, humangrowth hormone, insulin, bone growth factors, and other hormones).Furthermore, the device may include implantable drug delivery systemssuch as those disclosed in U.S. Pat. Nos. 3,773,919, 4,155,992,4,379,138, 4,130,639, 4,900,556, 4,186,189, 5,593,697, and 5,342,622which are incorporated in their entirety by reference herein.Implantable sensors for monitoring conditions such as blood pH, ionconcentration, metabolite levels, clinical chemistry analyses, oxygenconcentration, carbon dioxide concentration, pressure, and glucoselevels are known. Blood glucose levels, for example, may be monitoredusing optical sensors and electrochemical sensors. It should beunderstood that the implant devices may become embedded, or otherwiseintegrated, into the biological system.

In general, the material of the invention may be alternately formulatedto comprise, consist of, or consist essentially of, any appropriatecomponents herein disclosed. The material of the invention mayadditionally, or alternatively, be formulated so as to be devoid, orsubstantially free, of any components, materials, ingredients, factors,cellular constituents, cytokines, growth factors, tissue types, geneticelements, adjuvants or species used in the prior art compositions orthat are otherwise not necessary to the achievement of the functionand/or objectives of the present invention.

EXPERIMENTAL EXAMPLES

In one embodiment of the invention, a gene transfer system is includedwherein a genetically engineered cell suitable for use in the ATS isproduced. For example, as experimentally shown, a Rous Sarcoma VirusVector Model for Gene Transfer was created wherein a helper-independentretroviral vector, RCAS, derived from Rous Sarcoma Virus (RSV) was usedfor gene transfer in the in vitro and ex ova CAM model studies. A mouseVEGF gene (mVEGF), genebank number M25200, said genebank disclosureincorporated fully herein by reference and associated with the sequenceas shown in FIG. 22, was inserted into the RCAS proviral plasmid vectorin both “sense” and “antisense” orientations using standard recombinantDNA manipulations. Specifically, a 908 bp Taq I fragment containing themVEGF open reading frame was mobilized from pBSK+mVEGF and ligated intothe unique Cla I site of the RCAS-BP(A) proviral vector plasmid. Theligation products were screened by restriction mapping and both senseand anti-sense orientations were obtained. The resulting mVEGF andanti-mVEGF proviral DNAs were transfected into DF-1 chicken fibroblastcells using lipofectamine, and the cultures were passaged for two weeksto allow viral replication.

In one embodiment of the invention, evaluation of the in vitro impact ofthe RCAS viral vector on cell viability and morphology can be madewherein, as experimentally shown, once viral replication into DF-1 cellsis successful, the in vitro impact of the RCAS viral vector on viabilityand morphology of the chicken DF-1 cell line can be evaluated. TheRSV-derived RCAS vector efficiently infected DF-1 chicken fibroblastsand CAMs, and is easy to construct and propagate. FIGS. 2A to 2Edemonstrates that no effect on DF-1 cell viability and morphology wasnoted for any of the RCAS vectors used in these studies. The successfulinfection of the DF-1 cells was directly demonstrated using the GFP RCASvirus, which transformed the non-fluorescent DF-1 cells into fluorescentcells as shown in FIGS. 2D and 2E. Additionally, the presence of theRCAS virus in DF-1 cell cultures was demonstrated by immunocytochemistryusing an antibody to the Rous sarcoma virus p27 gag gene product (datanot shown) as well as soluble p27 viral coat protein.

Evaluation of Mouse mVEGF Protein Expression and RCAS Avian Leukosis(P27) Protein Expression after Viral Infection can be made in oneembodiment of the invention wherein after it is established that theviral infection did not impact cell viability or morphology, adetermination of the virus (p27) and mVEGF expression in the DF-1 celllines can be made. Experimentally, to determine and correlate mVEGFprotein and RCAS viral production over a one-week period, the variousinfected DF-1 cells were seeded at low confluence and monitored fortheir products until they reached confluence. For that, DF-1 cells andDF-1 cells previously infected with RCAS carrying gene for mVEGF,antisense-mVEGF (AS-mVEGF) or EGFP were seeded in triplicate in 12-wellplates at 1×10⁴ cells/well. The following days, an aliquot of theculture medium was taken out and replaced by fresh serum containingmedia. Aliquots were stored at −70° C. till evaluated for mVEGF and p27expression. Protein expression of mVEGF and p27 were measured by ELISAas described above. Measured for these studies was p27 antigen (a markerof virus content/production by cells) and mVEGF production by ELISA fromthe control DF-1 cells as well as the viral transfected cells usingELISA technology as shown in FIG. 3. All cell lines except thenon-infected parental DF-1 cells, produced significant amounts of p27antigen, as shown in FIG. 3A; (data not shown for DF-1, GFP:DF-1,AS-mVEGF:DF-1). The mVEGF production was detected only from RCAS-mVEGFtransfected DF-1 cells as shown in FIG. 3A and FIG. 3B. The peak mVEGFproduction by the mVEGF:DF-1 cells in vitro was 978±155 pg mVEGF/ml. Atime study of p27 and mVEGF expression in RCAS-mVEGF transfected DF-1cells (sub-confluent to confluent: FIG. 2A) indicated that both p27 andmVEGF production peaked at day 6 in culture (i.e. 80% confluent cells).Thus, our ELISA data clearly indicate that we can 1) infect DF-1 cellswith Rous Sarcoma Virus (anti-p27), 2) transfect mVEGF into these DF-1cells and 3) that these transfected cells clearly produce both RCASvirus and mVEGF.

In one embodiment of the invention a determination of cell viability andgrowth of cells incorporated into a matrix material composed primarilyof fibrin can be made. As experimentally shown a naturally occurringmatrix (fibrin) was used to investigate its utility to entrap cells andstill allow cell viability. In addition, the formation of fibrin clotsserved as a matrix to keep the cells localized, which is important forlater in vivo investigation where target gene delivery is an importantissue. Briefly, equal volumes of human fibrinogen (Fg) with varying Fgconcentration ranging from 6, 3 and 1 mg/ml (Sigma Chemical, St. Louis,Mo.) and cell suspension (2 million cells/ml) or media, were mixed and a50 μl aliquot was placed into the center of a 6-well petri-dish. 5 μl ofa 2.5E-3 U/μl thrombin solution (Sigma Chemical, St. Louis, Mo.) wasadded directly onto the fibrinogen/cell-mixture. Cells used in thesestudies included DF-1 cells, GFP-DF-1, AS-VEGF:DF-1 and VEGF:DF-1.Polymerization was complete within 15 minutes at 37° C. and produced athree-dimensional gel of fibrin entrapping cells or culture media in thecenter of the dish. As shown in FIG. 4 GFP:DF-1 cells are entrapped in afibrin clot. 3 ml of the culture media supplemented with Polybrene wereadded to each well after polymerization of fibrin. A 1 ml aliquot wastaken out of each well daily for a total of 10 days and replaced withfresh DF-1 culture media. Aliquots were stored at −70° C. till assayedby ELISA for p27 and VEGF expression as described earlier. Culturing thecells with varying concentrations of Fg was used to show that virus isreleased into the culture media and to determine fibrin clot stabilityover a 10-day culture period.

In one embodiment of the invention, the In Vitro Evaluation of VEGFProtein Expression and Avian Leucosis p27 Protein Expression Entrappedin a Fibrin Clot at various Fg Conditions can be made. As experimentallyshown, once fibrin successfully entraps cells over a 1-week time frame,as shown in FIG. 4, a determination of the virus (p27) and VEGFexpression in the DF-1 into the culture media incubated at various Fgcondition was made. As shown in FIG. 5A, VEGF:DF-1 cells entrapped in afibrin matrix with Fg concentration of 3 or 6 mg/ml expressed similarVEGF concentration. Fg concentration of 1 mg/ml had a slightly lowerVEGF expression when compared to Fg concentrations of 3 or 6 mg/ml.Control VEGF:DF-1 cells without addition of fibrinogen showedsignificant lower level of VEGF expression than cells embedded in fibrinmatrix. This indicates that the fibrin matrix is responsible inmodulating expression of VEGF. There was no VEGF expression detected inDF-1, GFP:DF-1 or AS-VEGF:DF-1 cell lines entrapped in the fibrin matrix(data not shown). All cell lines except the non-infected parental DF-1cells produced significant amounts of p27 antigen, which was notdependent on Fg concentration as shown in FIG. 5B (data not shown forDF-1, GFP:DF-1 and AS-VEGF:DF-1 cell lines). Thus, this ELISA dataclearly indicate that infected DF-1 cells entrapped in fibrin matrixwere able to express significant viral protein p27 and that VEGF:DF-1cells entrapped in fibrin matrix also produced significant levels ofVEGF.

A determination of the impact of thrombin on infected and parental DF-1cells can be made in one embodiment of the invention. For example, asexperimentally shown, once is shown that DF-1 infected cells entrappedin fibrin matrix express significant levels of p27 and VEGF in the caseof VEGF:DF-1 cells, the impact of Thrombin to DF-1 cells can bedetermined. For this, a Fg concentration of 3 mg/ml was used since itwas demonstrated that cells embedded in a fibrin matrix of 3 mg/ml Fgshowed a similar behavior as cells embedded in 6 mg/ml Fg. Cells wereprocessed as described above with regard to the determination of cellviability and growth of cells incorporated into a matrix material offibrin. However, an additional control was added. This additionalcontrol included cells treated with 5 μl of a 2.5E-3 U/μl thrombinsolution but no fibrinogen. As described above, a 1 ml aliquot was takenout of each well daily for a total of 6 days and replaced with freshDF-1 culture media. Aliquots were assayed by ELISA for p27 and VEGFexpression as described earlier.

In one embodiment of the invention, the in vitro evaluation of VEGFprotein expression and avian leucosis p27 protein expression entrappedin a fibrin clot at 3 mg/ml Fg can be made. As shown experimentally, afibrinogen concentration of 3 mg/ml and a thrombin addition of 5 μl of a2.5E-3 U/μl was used. As can be seen in FIG. 6A, VEGF expression ofVEGF:DF-1 cells entrapped in fibrin matrix was similar when compared tothrombin treated VEGF:DF-1 cells or control cells (VEGF:DF-1 cellswithout addition of thrombin and/or Fg). As expected, there was no VEGFexpression detected in DF-1, GFP:DF-1 or AS-VEGF:DF-1 cell lines eitherentrapped in the fibrin matrix or with thrombin addition (data notshown). All cell lines except the non-infected parental DF-1 cellsproduced significant amounts of p27 antigen, which was significantdifferent for Fg or thrombin condition as shown in FIG. 5B (data notshown for DF-1, GFP:DF-1 and AS-VEGF:DF-1 cell lines).

A determination of cell viability and growth incorporated into a matrixmaterial primarily composed of isolated basement membrane is included inone embodiment of the invention. Experimentally, it was previously shownthat a matrix material primarily composed of fibrin successfully entrapscells and that these cells were also able to release viral proteins. Anexperimental study was conducted to determine if the matrix Matrigel™was able to behave in a similar fashion as fibrin. Matrigel™, aspreviously discussed, is an isolated basement membrane obtained fromcells cultured in vitro, which has been used in a wide variety of invivo and in vitro studies of cell attachment, cell growth andangiogenesis. Like fibrin, Matrigel™ is a naturally occurring matrixderived from basement membrane that has a wide variety of binding sitesfor cells and factors (including growth factors and cytokines).Matrigel™ has been used extensively as a cell matrix/depot in a widevariety of in vitro and in vivo studies particularly in the area oftumor cell biology and angiogenesis. Matrigel™ is a liquid at 4° C. butbecomes a solid biological matrix when warmed to 37° C. This ability toconvert Matrigel™ from a liquid to a solid by simply raising thetemperature, allows for a wide variety of strategies for entrappinggenetically engineered cells, factors proteins and genes. Matrigel™ andother isolated basement membrane materials possess the characteristicsto serve as a tissue interactive biological matrix for the ATS. DF-1cells were utilized to determine the virus and VEGF expression in theDF-1 cell lines. Briefly, Matrigel™ and DF-1 cells (e, g. DF-1,GFP:DF-1, VEGF:DF-1) were mixed together at a ratio of 3:2 and 50 μl ofMatrigel™ cell suspension was pipetted into 6-well tissue culture plate.Wells were supplemented with culture media. An aliquot was taken out ofeach well daily for a total of 1 week and replaced with fresh DF-1culture media. Aliquots were stored at −70° C. till assayed by ELISA forp27 and VEGF expression as described earlier.

In one embodiment of the invention, after determining that Matrigel™successfully entraps cells the viral release of entrapped cells in aMatrigel™ clot can be determined. Experimentally the infectivity ofvirus carrying gene for GFP entrapped in Matrigel™ was determined. Forthis determination, 3E5 DF-1 chicken fibroblast cells were plated onto a6-well plate. Liquid Matrigel™ was mixed with GFP:RCAS viral supernatantat a ratio of 3:2 and 100 μl of Matrigel™/virus supernatant was placedin one well of a 6-well plate. As controls, 1000 of Matrigel™ was addedto wells of a 6-well plate. In addition, there was added 100 μl ofMatrigel™/virus supernatant to 0.7 cm² nylon fabric disks and placednylon containing Matrigel™/virus mixture into wells of a 6-well plate.Nylon fabric with and without addition of 100 μl Matrigel™ served asadditional controls in this study. In order to prevent polymerization ofMatrigel™ prior to placement into well, Matrigel™ was only handled withpipette tips kept on ice. Plates were placed in 37° C. incubator and 90%humidity and cells were inspected for green fluorescence daily. After afew days of incubation only DF-1 cells with viral addition showed greenfluorescence cells. Hence, virus entrapped in Matrigel™ is still able toinfect DF-1 cells.

In typical bioengineering applications, it is often desirable to markthe tissue site at which the cell factor production occurs. Therefore,in one embodiment of the invention, it is desirable to determine ifnylon fabric (mesh 100 μm pore size, Sefar America Inc., Depew, N.Y.) isusable as a cell carrier system. Specifically, for the cell carriersystem the nylon fabric was cut in 7 mm disks, ethylene oxidesterilized, dip coated in sterile egg white (EW), and placed into atissue culture treated 48-well-plate and manifested with an o-ring.Utilized were DF-1 chicken fibroblast infected with RCAS carrying genefor mVEGF, RCAS carrying gene for antisense to mVEGF, and RCAS carryinggene for EGFP. As an additional control also utilized were DF-1 chickenfibroblast only. Confluent monolayers were gently washed with phosphatebuffered saline (PBS), pH 7.2, and after a short exposure to trypsin,the cells were suspended in serum containing media. The cell suspensionwas counted in a hemocytometer and diluted with cell culture medium to afinal concentration of 5×10⁵ cells per ml. A 100 μl aliquot of that cellsuspension was added to the nylon fabric disk and the cells were allowedto grow on the nylon for several days. As an additional control, analiquot of cells was also added to nylon fabric disks, which were notdip-coated with EW. In order to determine cell density on nylon fabric,the nylon fabrics were transferred at day 4 post addition of cells totube containing fixative, washed repeatedly and stained withhaematoxylin solution. Excess haematoxylin stain was washed off andcells on nylons were observed for cell density. It was observed that ahigher number of cells grew on meshes previously dip-coated in egg whitecompared to meshes without egg-white coat. Furthermore, cells withaddition of egg white behaved similar when compared to cells grown inmedia without egg-white addition.

Experimentally, as discussed above, it was shown that DF-1 cells wereable to grow on protein treated nylon fabric. One embodiment of theinvention includes growing cells on Nafion® coated nylon fabric. Nafion®is a sulfonated tetrafluoroethylene based fluoropolymer-copolymer. TheNafion® fluoropolymer-copolymer is a material that is included in theouter layer of chemical- and bio-sensor described below. Experimentallynylon fabric disks, as described previously, were dip-coated withNafion® fluoropolymer-copolymer (Sigma) three times. After each coat adrying time at room temperature for a period of 10 minutes was observed.The Nafion® fluoropolymer-copolymer coated nylon disks were then placedinto an oven for 30 min at 120° C., nylon fabrics were washed 3-timeswith phosphate buffered saline and placed into a 48-well tissue cultureplate. Nafion® fluoropolymer-copolymer-treated nylon fabrics andnon-treated disks were secured at bottom of plate with the help of ano-ring (total of 3 nylon fabrics per condition). GFP:DF-1 cells at aconcentration of 5E4 cells per well were added to each well and platewas incubated at 37° C. and 90% humidity. Wells were inspected daily forcell survival and cell proliferation. Fibroblast adhered sparsely toboth Nafion® fluoropolymer-copolymer coated nylon fabric and non-coatednylon fabric in a similar pattern.

One embodiment of the invention includes adhesion of DF-1 cells toNafion® fluoropolymer-copolymer. Experimentally, Nafion®fluoropolymer-copolymer disks (50 mm diameter) were washed repeatedlywith dH₂O and soaked overnight in 0.9% sterile NaCl. Additional Nafion®fluoropolymer-copolymer disks were also dip-coated with Nafion®fluoropolymer-copolymer (Sigma) in order to confirm various Nafion®fluoropolymer-copolymer conditions used for sensor coatings. The diskswere cured at 120° C. for 30 minutes. After curing, the Nafion®fluoropolymer-copolymer disks were washed repeatedly with dH₂O. Nafion®fluoropolymer-copolymer disks (with and without EW coating) and Nafion®fluoropolymer-copolymer-coated disks (non-cured and cured disks with andwithout EW coating) were placed in 48-well plate and secured to bottomof well with o-ring (n=4 for each condition). Cells added to plain wellswith and without EW coating of tissue culture bottom served as positivecontrols. Cells were observed daily for cell survival, cell adherenceand cell proliferation. In general, cells grew much better on EW coateddisks when compared to non-coated EW disks with exception of Nafion®fluoropolymer-copolymer disks (non-cured). Here cells did neither adhereto EW coated nor non-EW coated Nafion® fluoropolymer-copolymer disks andtherefore did not survive. Cells on Nafion® fluoropolymer-copolymercoated Nafion® fluoropolymer-copolymer disks (cured) adhered onlysparsely to non-EW treated disks but these adhered cells were able toproliferate. Cells on Nafion® fluoropolymer-copolymer coated Nafion®fluoropolymer-copolymer disks (cured) treated with EW and Nafion®fluoropolymer-copolymer (cured) disks treated with EW showed a similarbehavior as control wells (wells without addition of disks but with andwithout addition of EW).

One embodiment of the invention includes adhering genetically engineeredcells and fibrin matrix systems to nylon. As discussed, it wasexperimentally demonstrated that growing cells on the nylon fabrics waspossible, therefore the approach of utilizing nylon fabrics as a carrierfor fibrin matrix and various cell lines was undertaken. Experimentallyutilized were 7 mm nylon fabric disks (foot mesh 35 and 1 μm pore size,Sefar America Inc., Depew, N.Y.) as a carrier for the cell-fibrinbiological matrix. Confluent monolayers of the various cell lines werewashed, trypsinized and re-suspended in serum containing media to afinal cell concentration of 2 million cells/ml. VEGF:DF-1 cells orcontrol cells (DF-1, GFP:DF-1) were mixed with equal volumes of aphysiological fibrinogen (Fg) solution (3 mg Fg/ml). Next, 50 μlaliquots of each of these solutions were placed onto the nylon diskswith the addition of 5 μl of a 2.5E-3 U/μl thrombin solution (SigmaChemical, St. Louis, Mo.). The nylon disk-cell-fibrin mixture was placedinto a tissue culture incubator at 37° C. and 90% humidity.Polymerization of fibrin matrix was usually completed within a fewminutes once placed into incubator. Cell adherence and cellproliferation of cell-fibrin matrix systems were investigated with alight microscope daily for up to one week. DF-1 cells and DF-1 cellsinfected with RCAS carrying genes for mVEGF or EGFP suspended in afibrin matrix and placed on the nylon disks, were all able to grow wellon the nylon fabric.

One embodiment of the invention includes preparation of an ex ova modelof the chick embryo. Experimentally, fertilized chicken eggs (WhiteLeghorn Strain) were obtained from the University of Connecticut PoultryFarm (SPAFAS, Storrs, Conn.). Initially, the fertilized eggs were placedhorizontally on trays and incubated at 38° C., with a relative humidityof 90% for 3 days. It should be noted that placing the eggs horizontallyduring the initial incubation period assures that the embryo develops inits proper position at the top of the egg. After the initial 3 days ofincubation the resulting eggs were used in the preparation of the ex ovaor shell-less embryo culture system. All steps in the preparation of theex ova model were conducted under aseptic conditions. Briefly, the3-day-old fertilized chicken eggs were first wiped with 70% ethanol andpermitted to air-dry to reduce contamination from the egg-shell surfaceduring the egg cracking procedure. The egg contents (embryo, yolk,albumin and chorioallantoic membrane (CAM)) were then directlytransferred into a 25×100-mm plastic petri-dish (Fisher Scientific,Pittsburgh, Pa.) by cracking the underside of the egg against a sharpedge. Transferring of the egg contents without damaging yolk or embryo,and positioning of the blastodisc uppermost and central in the petridish is critical in order to favor subsequent survival rate of theembryo. The resulting petri dishes, containing the chick embryo and CAM,were next placed back into a 38° C. incubator (with a relative humidityof 90%) for an additional 4 days (day 7 gestation). Generally, placementof various test materials onto the CAMs were conducted with chickembryos at gestation day 7, and test materials were simply placeddirectly on the CAM, and re-incubated at 38° C. and 90% humidity. Theresulting CAMs were evaluated for gross morphology and histology for upto 8 days post placement (day 15 of gestation).

To evaluate and document any gross morphologic changes induced in theCAM by various test materials including biomaterials, CAMs were examinedat various times post placement of the test material, using a Zeissstereo-microscope SR. The gross morphology of tissue reactions to thevarious test materials was documented with SPOT camera (DiagnosticInstruments, Inc., St. Sterling Heights, Mich.). In parallel studiesnon-treated chick embryos CAMs were also evaluated for gross morphology,and documented as described for the test material described above.Generally for these studies, evaluation of gross appearance of test andcontrol CAMs were done at 1 day, 4 days and 8 days post placement of thetest material on the CAM.

For histological evaluation of tissue reactions induced in the CAMs,control or test material treated CAMs were fixed in situ (10% bufferedformalin) at various days post placement of the test sample on the CAMs.The buffered formalin fixed tissue was then processed for paraffinembedding and sectioning. Generally, five μm sections were prepared ofthe various specimens, mounted on glass slides and stained withhematoxylin and eosin (H&E) for evaluation of histopathology. Histologicevaluation of tissue reactions in the CAMs was done on specimensobtained at 1 day, 4 days and 8 days post placement of the testmaterials.

Initially, the development of the chick embryo and CAM from day 3 to day17 of gestation. The initial development (i.e. days 1-3) of the chickembryo occurs in ova, prior to transferring the entire egg content intoa sterile petri-dish. Only egg contents without damage to the yolk orembryos, and with the position of blastodisc uppermost and central wereused in this study as shown in FIG. 7A. Usually about 80% of the 3 dayincubated eggs were successfully transferred to the petri dishes andinto the incubator. Typically the major lose of embryos occurs duringthe first three to four days after transfer to the petri dishes, andranges from 20-50%. Generally those embryos surviving the initial 3 daysex ova (day 6 gestation), have an excellent survival rate (i.e. up toalmost a 100%). At day 3 ex ova, the CAM is spread evenly over yolk andegg white layer. By this time-point the CAM has significant developmentof the vasculature. With continued incubation, the CAM and itsassociated vasculature, develops as a flat membrane, which reaches theedges of the dish by day 10 as shown in FIG. 7C. By gestation day 14areas of CAM are translucent due to reduction in yolk volume as shown inFIG. 7D. At gestation day 17 to 18 the vasculature is fully developed asshown in FIG. 7E. No difference in the CAM development is detected whencomparing in ova versus ex ova growth.

It should be noted that histologically the CAM, a transient respiratoryorgan for the developing chick embryo, consists of a mesodermal stromalined by an outer ectodermal (air side) and an inner endodermal composedof allantoic epithelium as shown in FIG. 8. The ectoderm layer iscomposed of a microvasculature, which serves primarily for gas exchange,and a chorionic epthelial layer. The mesodemal stroma is composed of acomplex vasculature supported by thin collagen fibers and fibroblasts.The ectoderm and endoderm are separated from the mesoderm by basementmembranes. The CAM has an approximate thickness of 100 to 200 μm.

In one embodiment of the invention chick ex ova CAM tissue, or othertissues, can be correlated to mammalian tissue with regard to similarreactions when stimulated with irritants or biomaterials. For example,experimentally, the CAMs were treated with a variety of substances andbiomaterials including: 1) bacterial endotoxins (lipopolysaccarides,LPS); 2) thread/suture; 3) nylon; and 4) silastic tubing (silicone).Acute inflammation was induced in the CAMs using endotoxin (LPS) afterplacement of the endotoxin on top of the CAM, and chronic inflammationwas induced using cotton thread, as discussed below. Finally, bothsilicone (silastic tubing) and nylon were also evaluated in the CAMs ofthe ex ova model, as discussed below.

Endotoxins are known to be potent inducers of acute inflammation in awide number of mammalian tissues, thus first determined was the abilityof endotoxins to induce acute inflammation in the ex ova CAM model. Toinduce acute inflammation in the CAM, 100 ng of an endotoxin fromEscherichia coli 0111:B4 (Sigma, St. Louis, Mo.) was placed on 7-day-oldembryo CAMs. For ease of visualization of the placement of the endotoxinon the CAM, the endotoxin was mixed with india ink. After various dayspost-placement of the endotoxin on the CAM, the CAMs were fixed with 10%buffered formalin in situ and processed for histology.

In order to evaluate the acute inflammatory response of the ex ova CAM,bacterial endotoxins were mixed with India ink, and was placed on top ofthe chorioallantoic membrane of 7-day-old embryos. The india ink wasadded to the endotoxin for ease of visualization over the 8 day timecourse of the study (days 1, 4 and 8 post placement). Gross morphologicevaluation of the CAMs demonstrated that post-placement of 1 to 8 daysresulted no detectable gross pathology as shown in FIGS. 9B and 9C. FIG.9A demonstrates the normal gross morphology of the CAM development of a12-day-old embryo. Evaluation of H&E stained CAM tissue sections fromday 1 post placement of endotoxins/india ink, demonstrated thatbacterial endotoxins induced a strong acute inflammatory response, withinflux of both plasma proteins (edema) and heterophiles (chickpolymorphonuclear leukocytes (PMNs)) into the CAM tissue. Heterophilesare the equivalent to mammalian neutrophiles, and are a principaleffector cell line of innate host defenses in avian. Generally, theacute inflammation induced by endotoxin/india ink remained localized onor near the surface of the ectoderm as shown in FIG. 9F. Hyperplasia ofthe ectodermal epithelial cells was all seen in the endotoxin/india inktreated CAMs. A thickening of the CAM occurred at sites of inflammationlikely due to tissue edema. The endotoxin/india ink treated CAMs werealso evaluated for tissue reactions after 4 days and 8 dayspost-placement of endotoxin/India ink. By 4 days post-placement of theendotoxin/india ink histologic evaluation of the CAMs indicated amassive influx of mononuclear leukocytes (monocytes and lymphocyte) intothe ectoderm layer of the CAM as shown in FIG. 9G. After 8 dayspost-placement of the endotoxin/India ink, the inflammation displayedsignificant resolution, as shown in FIG. 9H. By day 8 post-placement(PP), the inflammation appeared to be resolving on the surface of theCAM. The Histology of normal CAM is provided for comparison as shown inFIG. 9E.

Implants frequently induce chronic inflammation (foreign body reactions)when implanted in mammalian tissues. Cotton sutures represent a classicmodel of foreign body chronic inflammation, and were used to evaluatetissue reactions in the ex ova CAM model. For these studies, cottonthread fibers (0.5 cm to 1 cm in length) were prepared by unwindingstandard cotton thread and placed on top of the 7-day-old CAMs (3 dayincubation in the shell (in ova) plus 4 days ex ova). The resultingfibers were evaluated for gross and histologic changes as describedabove. Additionally, it was noted that the incorporation of threadfibers was accelerated when the thread fibers were pre-coated (dipped)in sterile egg white, prior to placement on the CAMs. Samples wereretrieved for gross and histological evaluation 1, 4 and 8 dayspost-placement of the thread fibers on the CAMs.

In the ex ova CAM model, tissue reactions to a “foreign object” (cottonthread fibers) was evaluated in the by implantation of cotton threadfibers on top of the CAM. It was demonstrated that egg white coatedthread fibers were rapidly incorporated into the ex ova CAMs as shown inFIGS. 10A-10H. Gross evaluation revealed that 1 day after depositionmost fibers were not significantly incorporated, but that thread fibersbegan to be incorporated 3 to 4 days post-placement as shown in FIGS.10B and 10C. By day 8 there was extensive incorporation of the threadfibers with association of pink haze surrounding the thread fiberssuggesting neovascularization around the incorporated fibers as shown inFIG. 10D. The gross morphology of the normal CAM of a 12-day-old chickembryo is presented in FIG. 10A. The resulting CAMs were next evaluatedfor histologic changes induced by the cotton fibers. At 1-daypost-placement of thread fibers, most of the cotton fibers were onsurface of CAM, with some incorporation of individual fibers as shown inFIG. 10F. Frequently, it was observed that chick PMNs (heterphiles)migrated from the microvasculature associated with the ectoderm, andsurrounded the individual fibers of the thread on the surface of theCAM. When individual fibers are incorporated at this stage, heterophilesand other inflammatory cells immediately surround those fibers. By 4 and8 day post-placement of the thread fibers an increase influx ofmononuclear leukocytes (macrophages and lymphocytes) was seen, withformation of giant cells surrounding the individual fibers, a hallmarkof chronic inflammation as shown in FIG. 10G-10H. Additionally, by 4days post-placement, areas of focal necrosis surrounding largeaggregates of cotton fibers were also seen, as well as fibroblast influxinto the inflamed tissue. At day 8 post-placement of the cotton fibersthere was continued presence of mononuclear leukocytes and giant cellssurrounding thread fibers. Additionally, a significant increase intissue necrosis, as well as the development of granulation tissue wasseen. This granulation tissue was characterized by neovascularization,fibroblast influx, and collagen deposition as shown in FIG. 10H. Thesestudies demonstrated that placement of an incompatible implant (i.e.cotton thread fibers) on the CAM induced not only acute inflammation,but also induced foreign body chronic inflammation and repair. Thus, itis clear that reactions seen in ex ova CAM model displays the sameclassic histologic hallmarks seen in foreign body induced chronicinflammation (i.e. macrophages, epithelial and giant cells) and repair(granulation tissue and fibrosis) of mammalian models.

In one embodiment of the invention the correlation between mammaliantissue and other tissue is determined with regard to medical gradenylon. Medical grade nylon is frequently used in implants, and generallyinduces minimal tissue reactions in mammalian tissues, thus we evaluatedthe reactivity of this biomaterial in the ex ova model. To test nylonreactivity in the ex ova model medical grade nylon screen (mesh 35 μmpore size, Sefar America Inc., Depew, N.Y.) was cut in 7 mm disks, steamsterilized, and dip coated in sterile egg white. The resulting nylondisks were then placed on the CAMs of 7-day-old chick embryos. After 1,4 and 8 days post-placement of the nylon disks, the CAMs were fixed insitu and the resulting specimens were processed as described above forevaluation of gross morphology and histology. Frequently when the nylondisks incorporated into the CAM were sectioned the nylon fractioned thetissue or the nylon fibers fell out of the tissue during embeddingprocessing. Although this did cause some loss of CAM tissue, routinelyadequate amounts of CAM remained in each section for evaluation. Thisexperimental study additionally showed that Egg white which containsglycoproteins and mucin excellerates the incorporation of biomaterials(nylon) into tissue.

Since a variety of biomaterials, such as nylon, are known to induceminimal tissue reactions when placed in mammalian tissues, the tissuereaction of the ex ova CAM to woven nylon fabric was evaluated. Grossmorphology of a nylon screen placed on top of the ex ova CAMs for 1, 4and 8 days can be seen in FIGS. 11A-11D. Evaluation of ex ova CAMsone-day post placement of the nylon screen indicated that there waslittle incorporation into the CAMs as shown in FIG. 11B. By 4 days postplacement of the nylon screen, the screen was significantly incorporatedwithin the CAM, and appeared as a translucent gel above the incorporatednylon screen disk as shown in FIG. 11C. After 8 days post-placement ofthe nylon screen, the nylon screen was almost totally incorporated asshown in FIG. 11D. For comparison, FIG. 11A shows a normal CAM of an11-day-old chick embryo. Histological evaluation of the tissue reactionsof the ex ova CAMs to the nylon screens, as shown in FIGS. 11E-11Hrevealed that 1 day post-placement the CAM surrounding the mesh showedno tissue pathology, likely due to the limited incorporation of nylondisk in the CAM. The nylon screen, which is denoted by “N”, can be seen“floating” (sectioning artifact) on top of the ex ova CAM as shown inFIG. 11F. When the nylon screen was placed on top of the CAM for 4 days,a moderate to significant incorporation was seen with no or littletissue reaction as shown in FIG. 11G. Day 8 post-placement showed asignificant incorporation of the nylon disk into the CAM as shown inFIG. 11H. Little or no inflammation was seen associated with theincorporated nylon disk. These studies clearly indicate that the ex ovaCAM reacts to nylon in a similar fashion as seen for tissue reactioninduced by nylon in mammalian tissues.

Silicone has frequently been used in implants, and generally has minimalreactivity in mammalian tissues. To evaluate silicone reactivity in ourex ova model, 0.5 cm to 1 cm long medical grade silastic tubing (DowCorning, Midland, Mich., 0.0301 cm ID×0.064 cm OD) were steam sterilizedprior to placement on 7-day-old CAMs. The silastic tubing was coatedprior to placement by dipping the tubing in egg white. At 1, 4 and 8days post-placement of the silastic tubing, specimens were fixed insitu. The resulting CAMs were evaluated for gross morphology as shown inFIGS. 12A-12D and histologically, as shown in FIGS. 12E-H.

As discussed, silicone has been used in bioimplants since it inducesminimal tissue reactions in mammals, thus the ex ova CAM for tissuereactivity to silicone tubing (i.e. silasic tubing) was evaluated. Atday 1-2 post placement of the silastic tubing significant incorporationof the CAM can be seen grossly, with in-growth of the CAM tissue,including the CAM vasculature into the lumen of the tubing as shown inFIG. 12B. By day 4 post-placement of the silastic tubing, the silastictubing showed consistent incorporation, with extensive in-growth of theCAM tissue and vasculature into the lumen of the tubing, as shown inFIG. 12C. Incorporation of the tubing continued at day 8 post-placementof the silastic tubing, as well as further in-growth of the CAM into thelumen, as shown in FIG. 12D. Histologic evaluation at CAM tissue day 1post placement of the silasic tubing, indicated that no significanttissue reactions occurred in the CAM tissue surrounding the silastictubing or in the CAM tissue and vasculature that had grown into thesilastic tubing, as shown in FIG. 12F. At day 4 post-placement, CAMtissue reactions to silastic tubing were insignificant as seenhistological, as shown in FIG. 12G. By day 8 post-placement of thesilastic tubing, the CAM tissue continued to display little or no tissuereactions to the silastic tubing, as shown in FIG. 12H. Additionally,the day 8 luminal in-growth of CAM tissue continued to appeared healthywith numerous blood vessels present. These studies clearly demonstratethat the ex ova CAM response to silicone is similar to that seen inmammalian tissue, and support the validity of the ex ova CAM as a modelof mammalian tissue reactions to biomaterials and bioimplants.

One embodiment of the invention includes the determination of RCAS viralinfectivity of CAM. Experimentally after determining an effective viralvector system, which promoted gene transfer in vitro, as discussedabove, the viruses were utilized to investigate their infectivity of theCAM of the chicken. Experimentally, utilized were DF-1 chickenfibroblast infected with RCAS carrying gene for mVEGF, RCAS carryinggene for antisense to mVEGF, and RCAS carrying gene for EGFP. As anadditional control also utilized was DF-1 chicken fibroblast only. Cellswere maintained in tissue culture flasks and once cells reached 80 to90% confluence, cell media was collected, filtered using a low proteinbinding membrane (25 μm pore size) and 50 μl of collected media wasadded to CAM of 7 or 8 day old chicken embryo. Experience in the CAMmodel was that direct placement of only the GFP:RCAS virus on the CAMresulted in few fluorescent cells appearing on the CAM. In parallelstudies using mVEGF:RCAS virus only, placement of the virus on the CAMresulted in little to no neovascularization. This may be due to the factthat viruses are very susceptible to drying effects.

One embodiment of the invention includes determining the infectivity ofCAM utilizing genetically engineered cells. For example, geneticallyengineered DF-1 Cells. As discussed above, it was experimentallydemonstrated that direct placement of the viral vector at the tissuesite resulted in relatively poor virus-gene incorporation. Thisdemonstration resulted in development and validation of a cell basedviral vector delivery system, e.g. the GFP:DF-1 and mVEGF:DF-1 cells.Experimentally utilized were DF-1 chicken fibroblast infected with RCAScarrying gene for mVEGF, RCAS carrying gene for antisense to mVEGF, andRCAS carrying gene for EGFP. As an additional control, also utilizedwere DF-1 chicken fibroblast only. Confluent monolayers were gentlywashed with phosphate buffered saline (PBS), pH 7.2, and after a shortexposure to trypsin, the cells were suspended in serum containing media.The cell suspension was counted in a hemocytometer and diluted with cellculture medium to a final concentration of 5×10⁵ cells per ml. Analiquot of that cell suspension was added to the CAMs of 7 or 8-day-oldchick embryos. After 4, 6 and 8 days post-placement of the celladdition, the CAMs were fixed in situ and the resulting specimens wereprocessed as described above for evaluation of gross morphology andhistology. Evaluation of the tissue demonstrated that direct placementof GFP:DF-1 cells on the CAM resulted in few fluorescent cells appearingon the CAM. In parallel studies using VEGF:DF-1 cells on the CAMresulted in little to no neovascularization. As was the case with onlyviral addition on the CAM, cells were also very susceptible to dryingeffects.

One embodiment of the present invention includes the preparation ofgenetically engineered cell-nylon systems for delivery of genes in theEx Ova model. In vivo production of heterologous proteins is often mostefficient when the tissue site is implanted with engineeredfactor-producing cells rather than viral vectors alone. In addition, inbioengineering applications, it would be desirable to mark the tissuesite at which the factor production occurs. To address these issuesexperimentally a nylon fabric (mesh 35 and 1 μm pore size, Sefar AmericaInc., Depew, N.Y.) was utilized as both a cell carrier system, as wellas a marker system, to track the site of cell transfer onto the CAM.Specifically, for the cell carrier system the nylon fabric was cut in 7mm disks, ethylene oxide sterilized, dip coated in sterile egg white,and placed into a tissue culture treated 48-well-plate and manifestedwith an o-ring. The cells were DF-1 chicken fibroblast infected withRCAS carrying gene for mVEGF, RCAS carrying gene for antisense to mVEGF,and RCAS carrying gene for EGFP. As an additional control, also utilizedwere DF-1 chicken fibroblast only. Confluent monolayers were gentlywashed with phosphate buffered saline (PBS), pH 7.2, and after a shortexposure to trypsin, the cells were suspended in serum containing media.The cell suspension was counted in a hemocytometer and diluted with cellculture medium to a final concentration of 5×10⁵ cells per ml. A 100 μlaliquot of that cell suspension was added to the nylon fabric disk andthe cells were allowed to grow on the nylon for 2 days prior toplacement onto the CAM. The nylon fabric disks were then gently dippedinto PBS prior to placement onto the CAMs of 8-day-old chick embryos.After 4, 6 and 8 days post-placement of the nylon fabric disks, the CAMswere fixed in situ and the resulting specimens were processed asdescribed above for evaluation of gross morphology and histology.

Since neither direct placement of viral vector alone nor geneticallyengineered cells alone placed onto the CAM, were able to significantlyinfect the CAM, a cell based viral vector nylon delivery system wasexperimentally developed. Initially utilized was the GFP-RCAS system torapidly evaluate gene transfer protocols for the CAM model. In order tomaximize GFP:DF-1 cell delivery/GFP expression to the CAMs, as well asdirectly mark the tissue site of cell implantation, a nylon fabric wasutilized as both a cell carrier/support system, as well as a markersystem, to track the site of cell transfer onto the CAM. For thesestudies in vitro cultured cells were grown on small circles of nylonfabric (“Disks”) and transferred those to the ex ova CAM membrane. FIG.13A demonstrates the brightfield image of GFP:DF-1 cells grown on nylondisks and placed onto the CAM at time point 0. FIG. 13B demonstrates thefluorescent image of GFP:DF-1 cells as the representing cell line toshow the ability of DF-1 cells to grow on nylon fabric. This studydemonstrated that the DF-1 cell line is able to grow on the nylonfabric. With reference to FIG. 14, it was also demonstrated that GFP:DF-1 cells grown on the nylon disks have the ability to infect the CAMtissue. It appears by comparing the brightfield images of the grosspictures shown in FIG. 14A-14C with the matching fluorescent images ofthe nylon disks shown in FIG. 14E-14G that only the CAM tissue growingon top of the nylon fabric is infected and as such expresses GFP. Itshould be noted that there was no difference in the chick development,morbidity or mortality rates in untreated control chick embryos whencompared to embryos treated with media only, DF-1 cells and GFP:DF-1cells producing GFP.

One embodiment of the present invention includes an alternative approachof adding the various cell lines directly onto the CAM. Experimentallynylon fabric was cut into rings, with an inner diameter of 7 mm, whereinthe nylon fabric was utilized as a marker for the cell application siteon the CAM. Confluent monolayers of the various cell lines wereprocessed as described above and added into the nylon ring.Additionally, the nylon ring was dip-coated in egg white prior toplacement onto the CAM. After 8 days post-placement of the nylon rings,the CAMs were fixed in situ and the resulting specimens were processedas described above for evaluation of gross morphology and histology.This study demonstrated that the DF-1 cells line is able to grow aroundnylon rings. The fluorescent ring seen in GFP:DF-1 nylon fabrics is aresult of cells accumulating at the edge of the inner ring as shown inFIG. 14H. FIG. 14D is the matching brightfield image to FIG. 14H. FIGS.14I-14L demonstrate the histology (H&E) of the nylon disks at timepoints 4, 6, and 8 days post-placement (PP) and nylon rings at day 8 PP.FIG. 14L demonstrates the histology (H&E) of nylon rings at day 8post-placement (PP). No histologic abnormality could be detected for anyof the various time-points.

One embodiment of the present invention includes the use of geneticallyengineered VEGF:DF-1 cell systems for delivery of genes in the Ex Ovamodel utilizing a nylon disk. Experimentally, the effect of mVEGFexpression in the ex ova CAM model was investigated by placingmVEGF:DF-1 cells directly on the CAM. As shown in the gross pictures ofFIGS. 15B to 15D, the use of nylon disks containing mVEGF-secreting DF-1cells resulted in a massive growth of vessels within the nylon fabric,which can be seen as early as 4 days PP. On day 4 PP an increase in thenumber of blood vessels in the stroma surrounding the ectoderm was seenhistologically as shown in FIG. 15F. The expansion of the vasculaturecontinued rapidly, and, by day 6 PP parts of the entire stroma werecovered with capillaries as shown in FIG. 15G. By day 8 PP, the entirestroma was filled with capillaries, pushing the nylon fabric towards theendoderm as shown in FIG. 15H. It should be noted that unlike mammalianred blood cells, avian red blood cells are nucleated and thus, can bemistaken for leukocytes. As controls for these studies, uninfected DF-1,AS-mVEGF or RCAS-GFP infected DF-1 cells were added to the CAMs. None ofthese control cells caused significant increase in blood vesselformation in the CAM. “Nylon-only” (i.e. nylon disks without any cells)controls were also utilized on the CAMs, which also did not induce newblood vessel formation. FIG. 15A shows a gross appearance of controlCAMs and FIG. 15E shows a histology of control CAMs. It should be notedthat the empty spaces in the histologic sections are where nylon fabricfibers, designed as N in FIGS. 15E to 15H and 16C and 16D, have fallenout of the tissue sections. This experimental study shows a method ofimplanting GE cells which produce VEGF which are attached tobiomaterials (Nylon), in a in vivo model ex ova CAM) these GE cells caninduce neovascularization at the sites of implantation, and alsodemonstrates the important of attachment of the cells for successfulgene therapy

One embodiment of the present invention includes the use of geneticallyengineered VEGF: DF-1 cell systems for delivery of genes in the Ex Ovamodel utilizing nylon rings. Experimentally nylon fabric was cut intorings, with an inner diameter of 7 mm, and utilized as a marker for thecell application site on the CAM. The cell addition to the nylon ringsutilizing the RCAS-mVEGF construct showed a ring of neovascularizationat the edge of the inner circle, which is a result of cells accumulatingat the edge of the inner circle, as demonstrated earlier by the use ofGFP:DF-1 cells as shown in FIG. 16B. In contrast, control cell additionfailed to induce neovascularization as shown in FIG. 16A. FIGS. 16C and16D show the histology of the nylon rings. As can be seen, an intensivecapillary development is the case for only the mVEGF:DF-1 cells but notfor the control. As discussed earlier, avian red blood cells arenucleated cells with red cytoplasm and should not be confused withleukocytes. Thus, it is experimentally established a simple, safe andefficient viral vector-cell delivery system for gene transfer in the exova CAM model. In addition, this experimental data clearly demonstratesthat the RCAS-mVEGF model can induce new vessel formation in the ex ovaCAM model. This experimental study shows another method for addinggenetically engineered cells to biomaterials, for example nylon disks,which can be used to enhance gene therapy, gene transfer, in vivo usingthe ex ova CAM model. It also shows how to evaluate the results of thegene therapy and demonstrates the evaluation of cellular attachment indetermining successful gene therapy.

One embodiment of the present invention includes mVEGF Expression inVivo. As previously shown experimentally the in vitro studiesdemonstrated that the mVEGF:DF-1 cells produced mVEGF. In vivoexpression of mVEGF in the CAM model was also experimentally carriedout. For these experiments, media, mVEGF:DF-1 cells or control cells(DF-1, GFP:DF-1 and mVEGF antisense:DF-1 cells) treated CAMs wereprepared using the nylon ring system as described above. At day 8 postimplantation the nylon rings were removed from the CAMs and the nylonring associated CAM tissue was removed and homogenized using a glasshomogenizer and a 0.1% triton PBS buffer to enhance protein extraction.The resulting homogenates were clarified by centrifugation, assayed formVEGF content by ELISA and for protein content by BCA protein assay(Pierce Chemical Company). All CAM data was normalized by calculation ofpg of mVEGF per mg of total protein. This experimental study shows amethod for the evaluation of the expression of a gene, for example, amouse gene (VEGF) in a biological model, for example, the Chicken Ex ovaCAM model.

Analysis of the CAM tissue homogenates demonstrated that CAM tissue fromthe mVEGF:DF-1 treated CAMs had significant mVEGF content (137.6±3.67pgs of mVEGF per mg total protein), that none of the CAM tissue frommedia or control DF-1 cells (DF-1, mVEGF antisense:DF-1 or GFP:DF-1Cells) had detectable mVEGF in the tissue homogenates. It should benoted that the ELISA detected only mouse mVEGF and not chicken mVEGF.Thus, these experimental studies are evaluating only the murine VEGFgene product expressed in DF-1 cells by gene transfer and show that themouse VEGF gene that was inside the genetically engineered cells usedfor this experimental gene therapy to induce new blood vessel wasexpressed in vivo in the chicken CAM, and that its expression was onlyseen when there was new blood vessel formation.

One embodiment of the present invention includes the use of fibrin forthe delivery of genes in the Ex Ova model. After experimentally showingthat fibrin clots are not only able to entrap cells but also to releasethe virus, as discussed above, the effect of neovascularization ofVEGF:DF-1 cells entrapped in a fibrin clot in the ex ova chick model wasdetermined. For this experimental study, VEGF:DF-1 cells or controlcells (DF-1, GFP:DF-1 and AS-VEGF:DF-1) (2 million cells/ml) were mixedwith equal amounts of a physiological Fibrinogen (Fg) solution (3 mg/ml)and a 50 μl aliquot was placed onto a nylon. After 5 μl of a 2.5E-3 U/μlthrombin solution (Sigma Chemical, St. Louis, Mo.) was added directlyonto the Fg/cell-nylon or Fg/media-nylon mixture, the variousnylon-disks were placed in a tissue culture incubator for about 15minutes at 37° C. for polymerization of the fibrin to occur. Disks werelifted out of the petri-dish, and care was taken to make sure most ofthe fibrin clot entrapping the cells or media was still attached to thenylon. The fibrin clot was then placed with the fibrin site down on topof the CAM of an 8-day old chicken embryo. Thus, it was experimentallyshown how to make an ATS which includes a matrix material and includesgenetically engineered cell(s). Also shown is how the ATS can be used inconjunction with a biomaterial support for such things a gene therapy,for example, for the induction of new blood vessels in vivo in the exova CAM model system.

As can be seen in the gross pictures of FIGS. 17B to 17D, the use offibrin matrix containing mVEGF-secreting DF-1 cells resulted in amassive growth of vessels within the nylon fabric, which can be seen asearly as 4 days PP. Control cells DF-1 as shown in FIG. 17A did notinduce any neovascularization on the CAM (data not shown for GFP:DF-1and AS-VEGF:DF-1). These experimental results show that an ATS whichincludes, for example, a genetically engineered cell with a matrixmaterial such as fibrin may be used in conjunction with a biomaterialsupport, such as nylon, to modify biological tissues, such as new bloodvessel formation in vitro.

One embodiment of the present invention includes the fabrication andutilization of Loop-Type Chemical Sensor. Experimentally, a chemical,acetaminophen, sensor, as shown in FIGS. 18 A and 18B was formed from aTeflon™ coated platinum (Pt) wire (Medwire, Mt. Vernon, N.Y.) which wascoiled around a 13 G needle after the Teflon™ had been removed at thewire extremities. The coiling area served as the working electrode forthe acetaminophen sensor. The coiled Pt wire was then anodized at 1.9 Vand cycled between −0.26 and +1.1 V vs a saturated calomel electrode andwith a Pt wire (Alfa Aesar, Ward Hill, Mass.) counter electrode in 0.5 MH₂SO₄ utilizing a CV-27 potentiostat (Bioanalytical Systems, WestLafayette, Ind.). Next, electrodeposition of a poly(o-phenylenediamine)(PPD) (Sigma Chemical, St. Louis, Mo.) film was conducted at +0.65 V for10 minutes. The sensor was dried for 0.5 h at room temperature beforedip coating the sensors with 6 layers of Nafion® fluoropolymer-copolymer(Sigma Chemical, St. Louis, Mo.). Sensors were cured for 0.5 h at 120°C. and stored dry at room temperature in closed containers. PPD filmformation and Nafion® fluoropolymer-copolymer coating were utilizedsince they are known to prevent or reduce biosensor fouling.

The performance of the sensor was evaluated in vitro as shown in FIGS.18C and 18D. Based on, for example, the nylon ring data, a circularsensor was chosen since it would act as a “corral” for the mVEGF:DF-1when added to the sensor placed on the CAM. The acetaminophen sensorperformance in vitro demonstrated that the sensor showed a dose dependresponse to acetaminophen over a range of 0.5 to 8 mM acetaminophen asshown in FIG. 18C. The slope for the response of the acetaminophensensors was 644 nA/mM, with an intercept of 390 nA (R²=0.988). The exova CAM model with an acetaminophen sensor in place at day 9 post-sensorplacement is presented in FIG. 18D. These in vitro studies demonstratedthe functionality and sensitivity of our sensor in vitro. This studyalso shows how to assemble an ATS-sensor combination wherein the ATSincludes a genetically engineered cell in addition to showing how todeploy the ATS-sensor in vitro implantation and testing offunction/lifespan in a biological model, for example, the ex ova CAMmodel

One embodiment of the present invention includes preparationsensor-Cell-Placement on CAM. Experimentally, after sterilizing thesensors by, for example, overnight UV exposure, sensors were dip-coatedin egg-white (EW) to enhance cell attachment. The sensors were placedinto a 60×15 mm tissue culture treated petri-dish and after the EW wasdried out, a fibrin clot containing either media or the cell suspensions(mVEGF:DF-1 or GFP:DF-1) was formed on top of the sensor loop. Theformation of fibrin clots served as a matrix material to keep the cellslocalized around the sensor. Briefly, equal amounts of human fibrinogen(6 mg/ml; Sigma Chemical, St. Louis, Mo.) and cell suspension (2 millioncells/ml) or media were mixed and a 50 μl aliquot was placed onto thesensor loop. 5 μl of a 2.5E-3 U/μl thrombin solution (Sigma Chemical,St. Louis, Mo.) was added directly onto the fibrinogen/cell orfibrinogen/media mixture. Polymerization was complete within 15 minutesat 37° C. and produced a three-dimensional gel of fibrin entrappingcells and sensor or culture media and sensor. Sensors were lifted out ofthe petri-dish, and care was taken to make sure most of the fibrin clotentrapping the cells was still attached to the sensor. The sensor wasthen placed on top of the CAM of an 8-day old chicken embryo.

Sensor performance in vivo was conducted 6 to 10 days post-placement ofthe sensor using the three-electrode system described above. Thepetri-dish containing the developing chicken embryo was placed into asand box, which was kept at 38° C. and a potential of 700 mV was appliedto the working electrode. After stabilization of the background current,200 μl of a 50 mM acetaminophen solution (in PBS) was injected i.v. andthe sensitivity to acetaminophen was determined and recorded onto achart recorder (Bioanalytical Systems (BAS), West Lafayette, Ind.). Thesensor current was monitored for approximately 20 minutes before anotheri.v. injection of acetaminophen was performed. Sensor response toacetaminophen was calculated as nano Amperes (nA) of the initial currentincrease. Thus, a method was experimentally shown for evaluating thefunction of a chemical sensor, for example an acetaminophen sensor andfor evaluation an ATS-sensor combination, wherein the ATS containsgenetically engineered cell(s) and a matrix material such as fibrin.

Impact of mVEGF Gene Transfer on Loop-Type Sensor Function in Vivo wasthen demonstrated experimentally. The focus of this experimental studywas to: 1) demonstrate the in vivo function of the acetaminophensensors, and 2) to determine the impact of mVEGF induced increase ofvessel density surrounding the sensor on sensor function in vivo. Ingeneral, sensors were incorporated after only a few days post-placementand only sensors completely incorporated were finally utilized tocompare responses between control sensors and angiogenesis inducedaround sensor. After 6-days to 10-days post-placement, sensors weretested using the 3 electrode system described in the methods sectionabove. Sensors implanted on CAMs with buffer or GFP:DF-1 cells displayedno induced neovascularization around the sensor as shown in FIG. 19. Inaddition, minimal sensor responses to i.v. acetaminophen injection weredetermined as shown in FIG. 20 wherein the results were media:133.33±27.64 nA (n=6); GFP:DF-1: 187.50±55.43 nA (n=6). In addition, itwas observed that the sensors implanted with mVEGF:DF-1 cells displayedmassive neovascularization as shown in FIG. 19B. Also observed was amassive sensor response to i.v. injected acetaminophen as shown in FIG.20 wherein the mVEGF:DF-1 results were 1387.50±276.42 nA (n=6)).Statistical analysis indicated that there was no statistical differencein sensor response between media treated sensors and sensors treatedwith GFP:DF-1 cells. However, when the responses of media treatedsensors or GFP:DF-1 treated sensors where compared to mVEGF:DF-1 treatedsensors there was major statistical significance (p<0.001). Asexperimentally has been shown, the VEGF-GE-Cell-ATS dramaticallyenhanced the in vivo function of the Chemical sensor in vivo, with thedramatic increase in sensor function being associated with the massiveneovascularization that occurred only at the site of theVEGF-GE-Cell-ATS-sensor implantation. Thus, ATS with genetic engineeredcell(s) and matrix material such as Fibrin enhance bothneovascularization and sensor function at sites of ATS-sensorimplantation.

Sensor function in the mVEGF:DF-1 and GFP:DF-1 treated sensor-CAMstudies were then compared over time as shown in FIG. 21. The control,GFP:DF-1 treated, sensors were functional at day 4 post-implantation butrapidly lost function by day 6 post-implantation as shown in the FIG. 21graph of control GFP:DF-1. This loss of sensor in the ex ova CAM modelgenerally paralleled the loss to sensor function seen in a wide varietyof mammalian models of implantable sensors. In contrast, the mVEGF:DF-1treated sensors were functionally similar to control GFP:DF-1 treatedsensors at day 4, but by day 6 post placement, displayed a massiveincrease in function, as shown in the FIG. 21 graph of the mVEGF:DF-1treated sensors. This increased function correlated with the appearanceof neovascularization around the sensor as shown in FIG. 19B. Clearly,increased vessel density surrounding a sensor in vivo does enhancesensor function in vivo, and provide “proof of principle” that genetransfer of angiogenic factors such as mVEGF can be used to enhancevessel density around implanted glucose sensors. Therefore, is has beenexperimentally shown that while a control, having a normal or non VEGFGE-cell containing ATS, will rapidly lose sensor functionally whenimplanted in vivo, a genetically engineered cell, such as a VEGF-GE-cellcontained in the ATS along with a chemical sensor will not lose functionand will even increase its function for sustained periods of time invivo.

Fabrication of Chemical Needle-Type Sensor is included in one embodimentof the present invention. Experimentally sensor fabrication of anacetaminophen sensor was performed wherein, the sensor comprising aTeflon™ coated platinum (Pt) wire (Medwire, Mt. Vernon, N.Y.), wascoiled after the Teflon™ had been removed at its extremities. Thecoiling area served as the working electrode for the acetaminophensensor. The coiled Pt wire was then anodized at 1.9 V and cycled between−0.26 and +1.1 V vs. a saturated calomel electrode and with a Pt foil(52 mesh, Alfa Aesar, Ward Hill, Mass.) counter electrode in 0.5 M H₂SO₄utilizing a CV-27 potentiostat (Bioanalytical Systems, West Lafayette,Ind.). Next, electrodeposition of a poly(o-phenylenediamine) (PPD)(Sigma Chemical, St. Louis, Mo.) film was conducted at +0.65 V for 10minutes. The sensor was dried for 0.5 h at room temperature before dipcoating the sensors with 6 layers of Nafion® fluoropolymer-copolymer(Sigma Chemical, St. Louis, Mo.). Sensors were cured for 0.5 h at 120°C. and stored dry at room temperature in closed containers. PPD filmformation and Nafion® fluoropolymer-copolymer coating were utilized toreduce or prevent biosensor fouling.

Prior to use of the acetaminophen needle-type sensor in vivo the sensorperformance was evaluated in vitro. The needle sensor configuration wasused since it resembles the same configuration as used for the glucosesensor discussed below. FIG. 23 shows the design of the acetaminophenneedle-type sensor. Briefly, acetaminophen sensor, working electrode,was characterized in pH 7.4 phosphate buffered saline at 0.7 V vs. asmall reference electrode (World Precision Instruments, Sarasota, Fla.)and utilizing a Pt foil as the counter electrode. The background currentwas allowed to stabilize for about 20 to 30 minutes, and increasingamounts of acetaminophen solution (Sigma Chemical, St. Louis, Mo.) wereadded to examine sensitivity and linearity. The acetaminophen sensorperformance in vitro demonstrated that the sensor showed a dose dependresponse to acetaminophen over a range of 0.5 to 8 mM acetaminophen. Theslope for the response of the acetaminophen sensors was 94 nA/mM, withan intercept of 57 nA (R²=0.991). These in vitro studies demonstratedthe functionality and sensitivity of our sensor in vitro, and thusallowed us to utilize these sensors for our in vivo studies, i.e. in theex ova CAM model.

Enhancing Acetaminophen Sensor Function Using Genetically EngineeredCells-Matrigel™-Systems to Induce Neovascularization in the Ex Ova CAMModel is included in one embodiment of the present invention.Experimental studies were conducted to determine the impact of mVEGFinduced increase of vessel density surrounding the sensor on needle-typechemical sensor function in vivo. Briefly, needle-type acetaminophensensors were prepared and processed as described above. Acetaminophensensors were placed into a microcentrifuge tube containing 40 μl of asuspension of Matrigel™ and cells at a ratio of 6:4 (15-20E4 cells pertube). Cells were submerged in cell DMEM media containing 10% fetalbovine serum. Sensors were carefully taken out of theMatrigel™/cell-media suspension or Matrigel™/media suspension to makesure that most of the suspension remains on the sensor. Sensors werethen placed on top of 7 or 8 day old chicken embryo and tested forfunctionality once incorporated into the tissue. An additional 30 μl ofMatrigel™ and cell suspension or media only was placed on top ofacetaminophen sensor once placed onto the CAM. In general, sensors wereincorporated after only a few days post-placement and only sensorscompletely incorporated were finally utilized to compare responsesbetween control sensors and angiogenesis induced around sensor.

FIG. 24 shows a summary of the in vivo acetaminophen sensor studiesperformed at 7 to 10 days post placement of the sensors onto the CAM.Sensors implanted on CAMs with mix of Matrigel™ and media (Media) orcontrol cells (DF-1 or GFP:DF-1) displayed no induced neovascularizationaround the sensor and had minimal sensor responses to intravenousdextrose injection as shown in FIG. 24 wherein the result were Media,16±14 nA (n=5); DF-1, 64±26 nA (n=5); GFP:DF-1 62±19 nA (n=5). Incontrast, the sensors implanted with mVEGF:DF-1 cells displayed massiveneovascularization, and equally massive sensor response to intravenouslyinjected dextrose as shown in FIG. 24 wherein the results for VEGF:DF-1was 293±21 nA (n=3). These studies clearly demonstrate that the uses ofthe VEGF-Matrigel™ system described here can dramatically enhance thefunction and lifespan of a chemical sensor, for example, anacetaminophen sensor in vivo.

One embodiment of the present invention includes the fabrication of abiosensor, for example, a glucose sensor. Experimentally a needle-typeglucose sensor, as shown in FIG. 25, having an outer diameter of 0.5 mmwas constructed from a platinum (Pt) wire and a silver (Ag) wire, withthe Teflon™ coating having been removed from the wire ends. The Pt wirewas coiled 10 times around the insulating Pt wire and served as theworking electrode. The Ag reference electrode wire was coiledapproximately 15 times around the Pt wire distal from the coiled Pt wirewith a 2 mm cap between them. After the probe was sonicated in water for5 min, silver chloride was then formed on the Ag wire electronically byapplying a current of 0.04 mA in a stirred 0.1 N HCl solution for 60minutes. Anodization and PPD film formation of the working electrode wasperformed as described for the acetaminophen sensors. Glucose oxidase(GOD) (EC 1.1.3.4, from Aspergillus niger, 158 U/mg) was immobilizedusing glutaraldehyde (aqueous 25%) as a cross-linking agent and bovineserum albumin as a carrier protein. 1 μl of that mixture was appliedthree times to the working electrode with a break of 30 minutes betweenthe single dipping procedures to allow the solution to dry on thesurfaces {Abel, 1999 #524}. After dip-coating the sensors with 6 layersof Nafion® fluoropolymer-copolymer, the sensors were cured for 0.5 hrs.at 120° C. and stored dry at room temperature in closed containers. Allchemical reagents were obtained from Sigma, St. Louis, Mo.

The in vitro sensitivity of each sensor to glucose was determined inphosphate buffered saline (PBS) by applying a potential of 700 mV versusAg/AgCl. After stabilization of the background current, glucose wasadded stepwise (final concentrations: 0, 4.8, 13.9, 22.6 and 30.9 mM) inorder to assess sensitivity and linearity. All testing was carried outat 37° C. Sensor output was measured at multiple glucose levels in orderto assess linearity. The sensor responds linearly to glucoseconcentration from 5 to 31 mmol/l (from 86 to 556 mg/dl). A typicalexample of the sensor response to increases in glucose concentration invitro is shown in FIG. 26 with a sensitivity of 15.5±3.8 nA/mM (n=38).These in vitro studies clearly demonstrated the functionality andsensitivity of the sensor in vitro, and thus allowed these sensors to beused for in vivo studies using the ex ova model.

In order to assess if Matrigel™ affects glucose sensor functionality invitro, sensors were tested with and without the matrix materialMatrigel™. Briefly, Nafion® fluoropolymer-copolymer needle sensors(outer diameter of 0.5 mm) were fabricated and validated as previouslydescribed. In vitro sensitivity of each sensor to glucose was determinedin phosphate buffered saline (PBS) by applying a potential of 700 mVversus Ag/AgCl. After stabilization of the background current, glucosewas added stepwise with final concentrations being 0, 4.8, 13.9, 22.6and 30.9 mM in order to assess sensitivity and linearity of the sensorresponse. All testing was carried out at 37° C. Matrigel™ was then addedto the working electrode of the glucose sensors and after polymerizationof the Matrigel™, the glucose sensors were tested in PBS as describedabove. Glucose sensor performance in vitro showed that sensors withoutaddition of Matrigel™ responded linearly to glucose concentration from 5to 31 mmol/l (from 86 to 556 mg/dl). Furthermore, addition of Matrigel™to the working electrode of glucose sensors did not affect sensorfunctionality and in some cases appeared to increase sensor sensitivity.These sensors had an average sensitivity of 10.6±3.2 nA/mM withoutMatrigel™ addition and an average sensitivity of 10.7±3.5 nA/mM with theaddition of Matrigel™ (n=4). These experimental in vitro studiesdemonstrated that matrix material, for example, Matrigel™ does notaffect the functionality and sensitivity of sensors in vitro, and thusallow the sensors to be used in in vivo studies. In addition, thesestudies identify a method to prepare and validate in vitro, a basementmembrane based ATS for implantable glucose sensors and that a basementmembrane based ATS does not interfere with the function of a glucosesensor in vitro.

One embodiment of the present invention includes enhancing glucosesensor function using and ATS which includes genetically engineeredcells-fibrin-matrix systems to induce neovascularization in an ex ovaCAM model. Experimentally Nafion® fluoropolymer-copolymer needle glucosesensors were fabricated, validated, implanted in the ex ova CAM's, andsensor function, as well as blood glucose levels were evaluated atvarious times after sensor placement. Experimentally determined waswhether the VEGF:DF-1 induced increase in vessel density would enhanceglucose sensor function in the ex ova CAM Model. GFP:DF-1-fibrin treatedsensors, lacking neovascularization, did not respond following an i. v.glucose injection (200 μl of 0.3 M glucose) (FIG. 26B), and theGFP:DF-1-fibrin treated sensor response did not correlate with directlymeasured blood glucose levels as shown in FIG. 27C. However, when aglucose solution (100 μl of a 0.3 M solution) was injected directly intothe tissue surrounding the sensor in the GFP:DF-1 treated CAM's, ahigh-magnitude signal (250 nA) was obtained, indicating that theimplanted sensor was still functional as shown in FIG. 27B. In contrast,the sensors in the VEGF:DF-1-fibrin treated CAM's retained theirfunction, as shown in FIG. 27E, and signals from the neovascularizedsensors correlated well with glucose concentration measurements madedirectly on contemporaneously sampled embryonic blood as shown in FIG.27F. Thus this experimental example teaches that using a fibrin-VEGF GEcell ATS system enhances the in vivo function of an implantable glucosebiosensor in the ex ova CAM model.

FIG. 28 shows a summary of the in vivo glucose sensor studies performedat 6 to 9 days post placement of the sensors onto the CAM. Sensorsimplanted on CAM's with protein coating (EW) (data not shown) or controlcells (DF-1 or GFP:DF-1) displayed no induced neovascularization aroundthe sensor and had minimal sensor responses to intravenous dextroseinjection, for example, FIG. 28 shows the results for EW, 0±0 nA (n=9);DF-1, 0±0 nA (n=6); GFP:DF-1 15.6±7.7 nA (n=9). In contrast, the sensorsimplanted with mVEGF:DF-1 cells displayed massive neovascularization asshown in FIG. 27D, and an equally massive sensor response tointravenously injected dextrose as shown in FIG. 28 where the resultwere mVEGF:DF-1, 75±8.6 nA (n=11). As can be seen in FIG. 28 addition ofDF-1 cells to the sensor implantation site resulted in no enhancement ofsensor function, when compared to the addition of a protein control(chicken egg white, EW). Addition of GFP:DF-1 cells to sites of glucosesensor implantation caused only a slight increase in sensor function.Addition of VEGF:DF-1 cells to sites of glucose sensor implantationcaused a massive and statistically significant increase in glucosesensor response compared do not only EW and control DF-1 cells, but alsowhen compared to the GFP:DF-1 cell treated sensor implantation sites.These studies clearly demonstrate that the uses of the VEGF-fibrinsystem described here can dramatically enhance glucose sensor functionand lifespan in vivo.

At 8-9 days post placement of the sensors, CAM tissue was fixed in situ(10% buffered formalin) and removed. The resulting fixed tissue,including the incorporated glucose sensor, was then processed andembedded in paraffin. Since glucose sensors are composed of silver andplatinum wire, removal of the sensor from the CAM tissue, prior tosectioning is required. Therefore, the sensor was carefully removed fromthe paraffin embedded tissue using fine tip forceps. Generally, removingthe sensor from the embedded tissue in a horizontal plane resulted inless tissue destruction. It should be noted that frequently fragments ofthe outer polymer layers of the sensors remained associated with theembedded tissue. Once the sensor was removed from the paraffin embeddedCAM tissue, the paraffin was melted, and the resulting “sensor free”tissue was re-embedded in paraffin. Generally, 5 μm sections wereprepared from the various CAM specimens, mounted on glass slides, andstained with hematoxylin-eosin (H&E) or trichrome stain (fibrosis) forevaluation of histopathology. This experimental details an example of amethod for processing tissue containing an ATS-glucose sensor.

Experimental histopathologic evaluation of the tissue reactions inducedin ex ova CAM tissue by GFP:DF-1 and VEGF:DF-1 fibrin gene deliverysystems were then conducted. Histological evaluation of both control(GFP:DF-1) and neovascularized CAM tissue clearly demonstrated thatextensive neovascularization occurred only in the VEGF:DF-1 treatedCAM's as shown in FIGS. 29A-29H. GFP:DF-1 treated CAM tissue generaldisplayed moderate inflammation, as shown in FIGS. 29A and 29E andfibrosis, as shown in FIGS. 29B and 29F. The histology for proteincoating (EW) and DF-1 cells was similar to that of the GFP:DF-1 treatedCAM's (data not shown). It should be noted that in the VEGF:DF-1 treatedCAM tissue, a variety of leukocytes were seen in association with theextensive neovascularization as shown in FIGS. 29C and 29G. Collagendeposition was also seen in association with the neovascularization asshown in FIGS. 29D and 29H. This experimental example demonstrates thatan ATS composed of VEGF-GE cells and basement membrane when implanted invivo can induce neovascularization.

One embodiment of the present invention includes enhancing glucosesensor function using genetically engineered cells-Matrigel™-Matrixsystems to induce neovascularization in the ex ova CAM model.Experimentally the impact on mVEGF induced vessel density on glucosesensor functionality in vivo was determined. Sensors were prepared andprocessed as described previously. Formation of bio-matrix was similaras described when using fibrin. Briefly, equal amounts of Matrigel™ (BDBiosciences, MA) and cell suspension (DF-1, GFP:DF-1 or VEGF:DF-1) ormedia were mixed in micro-centrifuge tubes and glucose sensorspreviously dip coated in egg white were submerged into theMatrigel™/cell mixture. Studies indicated that cell numbers ranging from1E5-2E5 gave comparable neovascularization, therefore 200,000 cells persite were utilized for the present experimental studies. Matrigel™polymerization was completed after exposure of the tube and sensor toroom temperature for a few minutes. The resulting sensor-Matrigel™-cellclots were released from the micro-centrifuge tube by the addition of0.9% NaCl, and placed on the CAM's of an 8-day chicken embryo. Anadditional Matrigel™-cell clot was transferred to the working electrodeof the glucose sensor prior to incubation of CAM's at 38° C. and 90%humidity. The Matrigel™ served as an ATS matrix material to: 1) preservecell viability by enhancing cell adherence; 2) enhance cell activation;and 3) to localize cells around the sensor. Sensors without the additionof either cell suspensions or media (EW coating only) served asadditional controls. Sensors were incubated for up to 7-10 days postplacement of the initially sensor-cell-Matrigel™ mixture. The CAM's wereevaluated for gross morphology and sensor function for up to 10 dayspost placement (day 18 of gestation).

FIG. 30 shows a summary of the in vivo glucose sensor studies performedat 7 to 10 days post placement of the sensors onto the CAM. Sensorsimplanted on CAM's with protein coating (EW) or control cells (DF-1 orGFP:DF-1) displayed no induced neovascularization around the sensor andhad minimal sensor responses to intravenous dextrose injection as shownin FIG. 30 where the results were EW, 0±0 nA (n=6); media, 0±0 nA (n=2);DF-1, 5±4.5 nA (n=6); GFP:DF-1 23.3±10.2 nA (n=6). In contrast, thesensors implanted with mVEGF:DF-1 cells displayed massiveneovascularization, and equally massive sensor response to intravenouslyinjected dextrose with the results being mVEGF:DF-1, 97.5±6.0 nA (n=6).As can be seen in FIG. 30 addition of DF-1 cells to the sensorimplantation site resulted in a minimal enhancement of sensor function,when compared to the addition of a protein control (chicken egg white,EW). Addition of GFP:DF-1 cells to sites of glucose sensor implantationcaused only a slight increase in sensor function. Addition of VEGF:DF-1cells to sites of glucose sensor implantation caused a massive andstatistically significant increase in glucose sensor response compareddo not only EW, media and control DF-1 cells, but also when compared tothe GFP:DF-1 cell treated sensor implantation sites. These studiesclearly demonstrate that the uses of the VEGF-basement membrane(Matrigel™) system described here can dramatically enhance glucosesensor function and lifespan in vivo.

One embodiment of the present invention includes a protocol for theimplantation of a glucose sensor into a mammal. Experimentally a murinemodel of implantable glucose sensors was developed utilizing 35-40 gmICR mice from Harlan (Indianapolis, Ind.). The mice were shaved at least24 hr pre-sensor implantation using a Wahl rechargeable electronicshaver. At the time of implantation, the shaved skin was disinfectedusing 70% isopropyl alcohol. For the present studies only one sensor permouse was implanted into the interscapular subcutaneous tissue of eachmouse. For the sensor implantation the mice were anesthetized with 1%isoflurane as approved by Animal Care at the University of Connecticut,Farmington Conn. Prior to sensor implantation, 0.1-0.150 ml ofinjectable sterile, pyrogen free, 0.9% NaCl was injected subcutaneously(s.q.) in the head-neck area of the anesthetized mouse to provide an“implantation pocket”. This implantation pocket was used to minimizetissue and sensor damage during sensor implantation. Next, a smallincision was made in the “implantation pocket” using corneal scissors,and the sensor was then implanted in the s.q. “pocket” with the twosensor leads exposed as shown in FIG. 31. The wound was closed with adrop of Nexaband Veterinary Surgical Glue (Chicago Ill.), and a smallpolyester mesh was placed on top of the exposed sensor leads as shown inFIG. 31. The sensor leads and the nylon mesh were secured to the shavedmouse skin by applying a coating of New “Skin First Aid and AntisepticLiquid Bandage” (Medtech Corp, Jackson Wyo.). Mice were kept underanesthesia until the New Skin Liquid Bandage dried. Drying wasaccelerated with the use of a hair dryer set at low temperature. The useof the nylon mesh and liquid bandage completely prevented removal of thesensors by the mice. Animals were observed until they recovered fromanesthesia. Once the sensors were implanted, mice were housedindividually, as a precaution to prevent dislodging of the sensor fromaggressive behavior between the mice when housed as a group. Dailyinspection of the sensor implantation site was necessary to prevent lossof mesh. All mice were maintained under specific pathogen-free (SPF)conditions, at the Animal Facility of the University of Connecticut,Farmington Conn., according to Animal Care Procedures. This experimentalexample demonstrates how to implant a sensor in the mouse in a fashionto minimize tissue trauma during sensor implantation as well as providean “in vivo pocket” for depositing ATS, drugs cells etc at the site ofsensor implantation also demonstrates the use of super glue to closeincision rather that suture thereby minimizing the tissue to the suture.Finally the experimental example demonstrates using nylon mesh and newskin to hold the mesh in place thereby to protecting the sensor leads sothey are not dislodged by the mouse, as well as providing antisepticcover for surgical site of implantation.

For the development of the murine model of implantable glucose sensors,an amperometric glucose sensor was used, the sensor functions based onthe detection of glucose using glucose oxidase. This sensor requiresexternal leads to allow for periodic evaluation of sensor function invivo. It should be noted that the present invention may include sensorswhich do not require external leads; for example, sensors having a radiofrequency connection to external instruments. Thus, when using a sensorwith external leads one of the key factors in the successful developmentof the implanted glucose sensor model was to develop techniques tomaintain placement and sterility of the implanted sensors in the mouse.To address these issues the sensor was implanted in the interscapularregion of the neck, which prevented the individual mouse from directlyremoving the implanted sensor as shown in FIG. 31. Initially, it wasfound that surgical glue could be used to close the surgical wound atthe implantation site, thus eliminating the need for sutures which inthemselves cause major tissue reaction as shown in FIG. 31A.Additionally, it was found that utilizing a small nylon mesh to coverthe exposed leads of the sensor, and an antiseptic coating polymer, i.e.New Skin®, both adhered the nylon mesh to the mouse skin and provided asterile antiseptic coating to protect the surgical wound from infectionas shown in FIG. 31B. Using this technique there was no damage to thesensor leads by the mouse, and no wound infection was observed as shownin FIG. 31C.

Determination of glucose sensor function in the mouse model was thenundertaken. At predetermined time point's post-sensor implantation,sensor function and blood glucose levels were assessed. For thesestudies animals were anesthetized with 1% isofluorane, and weremaintained at 37° C. using a heating blanket. The nylon mesh, and theNew Skin® Liquid Bandage were removed with acetone prior to sensortesting. The exposed sensor leads were then connected to a potentiostat(working potential: 700 mV) for amperometric measurements. Current wascontinuously recorded during the experiment until stable (baseline)current was achieved. To elevate blood glucose levels in these animalsthey were given 80-150 μl of a 0.5 g/ml dextrose solutionintraperitoneally (IP) after establishing sensor baseline period ofusually 20 to 30 minutes. To monitor blood glucose levels, a single dropof blood was obtained from the tail vein before glucose injection(baseline) and at 5, 10, and 20 minutes post glucose injection. Furtherglucose measurements were then made every 10 minutes until theconclusion of the experiment. All blood glucose levels were determinedusing an OneTouch Ultra® glucose meter (Lifescan, Johnson & Johnson).Sensor performance was followed for about 40 to 60 minutes initially,and for at least 20 to 30 minutes at each subsequent testing session.

In one experimental the glucose sensor function was tested in each mouseimmediately following implantation, designated as 1-hour postimplantation (HPI), 5 HPI, 1 day post implantation (DPI), 2 DPI, 3 DPI,7 DPI, 14 DPI and occasionally up to 30 DPI. Between the sensor functiontests, the mice were left unrestrained in their cages, withoutpolarization of the sensor. A total of 24 mice were tested, eachimplanted with a different sensor. Sensor sensitivity was calculated bya two-point calibration wherein one point was taken at baseline and onepoint was taken at the peak glucose.

To experimentally evaluate sensor function in the mouse model, sensorfunction in the mouse was analyzed for 1 HPI and up to 30 DPI. In thesestudies, glucose blood levels parallel sensor function at both 1 and 5hours post sensor implantation as shown in FIG. 32 where the results areshown for 1 hr and 5 hr. Analysis of blood glucose and sensor functionat 1-day post sensor implantation clearly indicated that although theblood glucose levels were elevated by i. p. injection of the glucose,that there was little or no response from the implanted sensors as shownin FIG. 32 at the 1 d point. Analysis of the blood glucose and sensorfunction at 2, 3, 7, 14 and 30 days post sensor implantationconsistently demonstrated that although blood glucose levels wereelevated by i. p. injection of glucose, there was no sensor function asshown in FIG. 32 at the 2 d, 3 d, 7 d, 14 d, and 30 d points. This rapidloss of sensor sensitivity, as shown in FIG. 33, and the loss of sensorfunction, as shown in FIG. 34, within the first day post implantationwas seen in this sensor implant arrangement in the mouse model. Thisloss of sensor function is highly correlated to the general pattern ofloss of sensitivity and sensor function seen in other animal models andin man. For example, this experimentally shows that the loss of sensorfunction seen in mice is similar to the loss of glucose sensor functionseen when glucose sensors are implanted into human skin.

Sites of sensor implantation were evaluated grossly for redness,swelling, warmth or other signs of inflammation both before and afterremoval of the protective nylon mesh. To evaluate the tissue responsesto implantation of the glucose sensor at various time points, individualmice were euthanized, and tissue containing the implanted sensors wasremoved and fixed in 10% buffered formalin. Next, the buffered formalinfixed tissue was then paraffin embedded. Prior to sectioning theembedded tissue, the sensor was removed from the paraffin embeddedtissue. Removing the sensor, while it was embedded in the paraffin,resulted in minimum tissue damage as a result of sensor removal. Afterremoval of the sensor, the tissue was re-embedded, and processed forsectioning in a manner similar to that described in the CAM model. Theresulting tissue sections were processed using hematoxylin and eosin(H&E), as well as trichrome staining techniques. Histopathologicevaluation of tissue reactions at sites of sensor implantation was doneon mouse specimens obtained at 1 day, 3 days, 7 days, 14 days, and1-month post implantation of the glucose sensor. The tissues from thesites of sensor implantation were examined for evidence of loss of celland tissue architecture, acute and chronic inflammation including giantcell formation, necrosis, as well as fibrosis and vessel regression.

A determination of the acute and chronic tissue responses to theimplanted glucose sensors in the mouse model was also undertaken. Thenature of the tissue reactions induced in the mouse by the implantedglucose sensors was determined, by analyzing tissue specimens obtainedfrom sites of sensor implantation at the 1 day to 30 days post sensorimplantation. The analysis focused primarily on the tissue reactionssurrounding the working electrode (WE) (which contains glucose oxidase)and the reference electrode (RE), which lacks glucose oxidase but doesrelease silver chloride, for example, as shown in FIG. 25. The “bridge”wire between the WE and RE displayed significantly less tissue reactionwhen compared to the highly tissue reactive reactive WE and themoderately tissue reactive RE.

Histopathologic analysis of the tissue surrounding the WE indicated thatat day 1 there was a moderate diffuse inflammatory process,characterized by necrosis, edema, fibrin deposition and the presence ofboth polymorphonuclear leukocytes (PMN's) and monocytes as shown inFIGS. 35A and 35B. By 3 DPI, the sensor was surrounded by dense band ofinflammatory cells with some necrosis surrounding the implanted sensoras shown in FIGS. 35C and 35D. The inflammatory cells were primarilyPMN's and macrophages. Adjacent to the dense inflammatory band, adiffuse region of inflammation characterized by numerous activatedmacrophages and fibroblasts with occasional lymphocytes was seen asshown in FIG. 35C. Initial collagen deposition was seen distal to theband of inflammatory cells surrounding the implanted glucose sensor asshown in FIG. 35D. By day 7 and 14 post sensor implantation the tissuereactions were characterized by the presence of large numbers ofmacrophages and activated fibroblasts surrounding the working electrodeas shown in FIGS. 35E, 35H, and 35G. It was also noted that there wassignificant neovascularization of the tissue adjoining the workingelectrode. Giant cell formation particularly around the workingelectrode as early as 7 DPI was also observed as shown in FIG. 35.Trichrome staining of the day 7 and 14 tissue surrounding the workingelectrode demonstrated the presence of numerous activated fibroblastsassociated with growing ribbons of collagen surrounding the workingelectrode as shown in FIGS. 35F and 35H. By day 30 the WE was surroundedby a dense band of inflammatory cells and activated fibroblasts as shownin FIGS. 37A and 37B, intermingled with a dense band of collagen asshown in FIGS. 37E and 37G. Thus, these experimental example shows thattissue reactions at sites of glucose sensor implantation in mouse skininclude those due to the glucose oxides containing region of the sensorwhich function to induce sever tissue reactions that are associated withthe loss of sensor function.

Experimentally it was observed that significantly less inflammatoryreactions occurred in the tissue surrounding the reference electrode aswell as in the tissues surrounding the Teflon-coated wire, which joinsthe working electrode with the reference electrode as shown in FIGS. 35and 36. At day 1 post implantation the tissue surrounding the referenceelectrode had light diffuse inflammation, characterized by the presenceof PMN's and monocytes as well as edema and some fibrin deposition asshown in FIGS. 35I and 36J. It was noteworthy that there was relativelylittle tissue necrosis immediately surrounding the reference electrode,compared to what was seen surrounding the working electrode. At 3 dayspost sensor implantation, inflammation characterized by PMN's andmacrophages was still present, but was localized close to the surface ofthe reference electrode as shown in FIGS. 35K and 35L. The inflammatoryreactions did not extend significantly into the tissue adjoining thereference electrode as shown in FIG. 35K. Only wispy collagen fiberswere seen in the tissue next to the inflammation as shown in FIG. 35L.At days 7 and 14 a low-grade inflammation, characterized by the presenceof PMN's and macrophages, continued in close proximity to the surface ofthe reference electrode as shown in FIGS. 35M, 35N, 35O and 35P. Therewas an increased appearance of activated fibroblasts and loose collagenband formation as shown in FIGS. 35N and 35P. Neovascularization waslimited and sparse in the tissue surrounding the reference electrode asshown in FIGS. 35M and 35O. By 30 days post sensor implantation, theinflammatory reactions were still seen in close proximity to the surfaceof the reference electrode, with an increasingly dense layer of collagenseen bordering the inflammation as shown in FIGS. 37D and 37H. Althoughneovascularization was seen distal to the reference electrode it wasdramatically less frequent and dense, compared to what was seen at theworking electrode.

Experimental determination of glucose sensor functionality in vitro withvarying glucose oxidase levels was undertaken. Glucose oxidase is knownto cause tissue damage. Therefore, glucose sensors were prepared withvarying concentration of glucose oxidase and its affect on sensorfunctionality and sensitivity was investigated. Briefly, glucose oxidase(20 mg/ml) at a serial dilution of 1:10 was immobilized usingglutaraldehyde as a cross-linking agent and bovine serum albumin as acarrier protein. 1 μl of that mixture was applied three times to theworking electrode with a break of 30 minutes between the single dippingprocedures to allow the solution to dry on the surfaces. Afterdip-coating the sensors with 6 layers of Nafion®fluoropolymer-copolymer, they were cured for 0.5 hrs. at 120° C. andstored dry at room temperature in closed containers. Sensorfunctionality was determined in PBS and with addition of glucose asdescribed above. No sensor response was detected for glucose sensor witha glucose oxidase dilution of 1/1000. Glucose sensor with a glucoseoxidase dilution of 1/10 of initial concentration showed a loss ofsensor sensitivity of 50%. The lowest detectable sensitivity for glucosewas detected in glucose sensors with a glucose oxidase dilution of 1/100(1.8E-3 mg/ml GO). Since it had been demonstrated earlier that GO isextremely tissue toxic it was concluded that a GO concentration of 6mg/ml (1.8E-2 mg/ml GO per sensor) is sufficient to reliable detectglucose levels in vivo. Experimentally this shows that the levels ofglucose oxidase in a glucose biosensor affects the in vitro function ofthe sensor, and demonstrates that the minimum levels of glucose oxidaselevel required for detectable sensor function is about 0.6 mg/ml.

Since the in vitro data demonstrated that glucose oxidase (GO) is toxicto cells, it was investigated whether glucose sensors with a lesseramount of GO would reduce inflammation around the tissue of implantedglucose sensor. For this study, glucose sensors with various GOconcentrations (3 consecutive serial 1/10 dilution's as described above)were implanted into the mouse model following the implantation protocolas described previously. Glucose sensor functionality and histologicevaluation was also conducted as described previously. Unexpectedly,glucose sensors with less GO showed same failure as non-diluted GOglucose sensors. However, histologic evaluation showed that tissueresponses to glucose sensors with diluted GO had significantly lessinflammation than glucose sensors with full concentration of GO.

One embodiment of the present invention includes an ATS-glucose sensorimplanted in a mammal. It has been experimentally demonstratedpreviously that glucose sensors with a diluted concentration of glucoseoxidase showed a glucose sensor response similar to non-diluted GOglucose sensor but showed less severe inflammation around theimplantation side. Therefore, it was investigated as to whether aglucose sensor with ATS showed a better sensor lifetime. Briefly,glucose sensors ( 1/10 diluted GO) were dip-coated with Matrigel™ andwere implanted into mice using procedures described previously. However,prior to closing wound side, 100 μl of Matrigel™/saline (3:2 ratio) wasinjected around glucose sensor side. Control mice received no Matrigel™treatment. Glucose sensor function as described above was tested in eachmouse immediately following implantation, designated as 1-hour postimplantation (HPI), 5 HPI, 1-day post implantation (DPI), 2 DPI, 3 DPI,and 6 DPI. Between the sensor function tests, the mice were leftunrestrained in their cages, without polarization of the sensor. Sensorsensitivity was calculated by a two-point calibration method wherein onepoint is taken at baseline and one point is taken at the peak glucose.

To evaluate sensor function in our control mice and Matrigel™ treatedmice, sensor function in the mouse was analyzed for between 1 HPI and 6DPI. In these studies, glucose blood levels parallel sensor function atboth 1 and 5 hours post sensor implantation. Analysis of blood glucoseand sensor function at 1-day post sensor implantation clearly indicatedthat although the blood glucose levels were elevated by i. p. injectionof the glucose, that there was little or no response from the implantedsensors in the control mouse. Analysis of the blood glucose and sensorfunction at 2, 3, and 6 days post sensor implantation consistentlydemonstrated that although blood glucose levels were elevated by i. p.injection of glucose, there was no sensor function in the control mouseas shown in FIG. 38. However, glucose sensor functionality in Matrigel™glucose sensor treated mouse paralleled blood glucose levels. Thisbehavior was consistent for time-points 1 HPI, 5 HPI, 1 DPI, 2 DPI and 3DPI. Sensor sensitivity was in the range of under 10 to about 13 nA/mMfor Matrigel™/sensor treated mouse at 1 DPI, 2 DPI and 3 DPI, as shownin FIG. 38. Thus, an ATS, such as Matrigel™ around a glucose sensorincreases the lifetime of an implantable glucose sensor.

One embodiment of the present invention includes an implanted glucosesensor in a dexamethasone treated mouse model. Experimental glucosesensor implantation in dexamethasone treated mice was conducted the sameway as described for control mice above. However, mice were treated withdexamethasone i. p. 24 hrs and 1 hr prior to sensor implantation.Control mice were not treated with dexamethasone at any giventime-point. Mice were injected with dexamethasone i. p. on a dailybasis. Daily inspection of the sensor implantation site was necessary toprevent loss of mesh. All mice were maintained under specificpathogen-free (SPF) conditions, at the Animal Facility of the Universityof Connecticut, Farmington Conn., according to Animal Care Procedures.

Evaluation of sensor function in the mouse model was accomplished byanalyzing sensor function in control mice and in the dexamethasonetreated mice for 1 HPI and up to 8 DPI. In these studies, glucose bloodlevels parallel sensor function was monitored at both 1 and 5 hours postsensor implantation, as shown in FIG. 39 (4 hr. and 8 hr.), for bothcontrol mice, having no dexamethasone treatment, and dexamethasonetreated mice. Analysis of the blood glucose and sensor function at 1 daypost sensor implantation in the control mouse clearly indicated thatalthough the blood glucose levels were elevated by I. p. injection ofthe glucose, that there was little or no response from the implantedsensors in the control mice as shown in FIG. 39 (1 d). However, analysisof the dexamethasone mouse showed glucose levels paralleled sensorfunction. Analysis of the blood glucose and sensor function at 3, and 8days post sensor implantation in the control mouse consistentlydemonstrated that although blood glucose levels were elevated by I. p.injection of glucose, there was no sensor function as shown in FIG. 39(3 d and 8 d). This rapid loss of sensor sensitivity and sensor functionwithin the first day post implantation was seen in virtually all sensorstested in the control mouse model. Furthermore, the general pattern ofloss of sensitivity and sensor function seen in our control mice wassimilar to that seen in both other animal models and man. On the otherhand, glucose sensors implanted into mice treated with dexamethasone I.p. showed blood glucose levels paralleling sensor functionality. Thus,by controlling inflammation around an implanted glucose sensor it ispossible to extend the lifetime and functionality of a glucose sensor.This experimentally demonstrates that systemic administration ofdexamethasone dramatically enhance the function of a glucose sensor whenimplanted in the skin of mice.

One embodiment of the present invention includes a matrix materialangiogenesis assay in a mammalian model. As previously shownexperimentally, the gene transfer in the CAM model is used as a screento establish “proof of principle” the concepts and tools (candidategenes) that are successful in the ex ova model need to be evaluated inmammalian models. A simple first step in this evaluation is to implantthe chicken RCAS/DF-1 cells+/− the VEGF gene in immunodeficient mice(designated nu/nu). Immunodeficient are routinely used to grow cellsfrom other species in vivo. Briefly, chicken VEGF:DF-1 and AS-VEGF:DF-1(VEGF anti-sense gene control) cells were trypsinized to free fromplate, centrifuged and re-suspended carefully with a 25 ml pipette in 20ml serum-free media. An aliquot was removed and cells were counted witha hemacytometer. Cells were centrifuged again and re-suspended in 10 mlof serum-free media. For five animals 5×10⁶ cells were placed into a 5ml snap cap centrifuge tube. Cells were centrifuged and re-suspended in0.3 ml serum-free media with the addition of 0.7 ml Matrigel™. Animalswere anesthetized and each animal received 200 μl of Matrigel™/cellsuspension on each side midway between flank and shoulder blades. As acontrol, cells without Matrigel™ were also injected into the skin of themice at separate skins sites. Harvest was conducted after 8-days postinjection by CO² asphyxiation and skin dissection to reveal matrigelpellet. Pellets were collected with the overlying skin and placed infixative for 2 hours and then transferred to PBS for 3-4 hours followedby 70% ETOH and storage at 4° C. until processing for paraffin sections.Tissue samples were also obtained from the skin sites, which receivedcells without matrigel. The resulting tissue reactions were evaluatedhistologically. This experimental example teaches a method to constructand implant an ATS system composed of genetically engineered cells(chicken cells which overproduce the angiogenic factor VEGF) into animmunodefiecient mouse (nude/nude) in a Matrigel™ to determine 1) theability of this ATS to induce neovascularization in the mouse model and2) to protect the genetically engineered cells from destruction bytissue reaction i.e. inflammation.

One embodiment of the present invention includes using VEGF:DF-1 toinduce neovascularization in immunodeficient mice (nude/nude).Experimentally In order to determine if chicken VEGF:DF-1 cellsentrapped in ATS system (Matrigel™) are capable of inducingneovascularization in mice, VEGF:DF-1 cells were suspended in Matrigel™and injected s.q. in the back of the mice. Mice were sacrificed 8 dayslater, and the injected tissue was removed, fixed and processed for H&Estaining. Mice injected with DF-1/antisence VEGF(AS-VEGF:DF-1) cells(controls), displayed no neovascularization around the Matrigel™ asshown in FIG. 41B. In contrast, mice injected with VEGF:DF-1 cellsdisplayed a robust neovascularization around the Matrigel™ as shown inFIG. 41A), and in many cases the neovascularization penetrated theinterior of the Matrigel™ as shown in FIG. 41C, note that the arrowspoint to microvessels and indicate a colony of VEGF:DF-1 cells. It wasalso noted that tissue inflammation was minimal around the implantedATS, and when present it did occur at the margins of the ATS, it did notpenetrate into the ATS. This ability of the ATS system to 1) causelittle tissue inflammation when implanted in vivo and 2) to block accessof the inflammatory cells (i.e. inflammatory cells of innate immunitysuch as PMNs and macrophages) to the engineered cells in the ATS,resulted in protection of the cells within the ATS and enhancements ofcell viability and function in vivo. Finally, it was observed that whenthe genetically engineered VEGF-DF-1 cells where injected into the skinof the mice without Matrigel™ the cells failed to induce anyneovascularization. This clearly indicates the critical role of theMatrigel™ basement membrane in both promoting and protecting cellsimplanted at tissue sites, and underscores the importance of the matrixin both protecting the sensor as well as promoting cell survival andfunction at the site of ATS-sensor implantation. This experimentalexample shows that an ATS system composed of genetically engineeredcells (chicken cells which overproduce the angiogenic factor VEGF) intoan immunodeficient mouse (nude/nude) in a Matrigel™; this ATS systeminduces neovascularization into the ATS system only when the cellsengineered to overproduce VEGF are in the ATS but not when control (VEGFanti-sense) engineered cells are in the ATS. Additionally, thisexperimental example demonstrates that the ATS induced minimal tissueinflammation when implanted and that basement membrane matrix(Matrigel™) appears to protect the engineered cells from destruction bythe tissue inflammation, because the cell within the Matrigel™ wereviable. Finally, this experimental example shows that Matrigel™ ATS isrequired to promote efficient neovasculariztion; e. g. cells injectedinto the skin of the mice without matrigel did not induceneovascularization, thus indicating the important role of the matrixmaterial, for example basement membranes, in the ATS in cell viabilityin vivo.

One embodiment of the present invention includes the use of an ATS whichincludes Matrigel™, or other matrix material, in combination withvarious cells and a sensor. For example: Matrigel™+normal vascular stemcells+sensors; (Matrigel™+cytokines bound to matrigel)+normal vascularstem cells+sensors; Matrigel™+normal vascular stem cells+engineeredsupport cells+sensors; Matrigel™+normal vascular stem cells+engineeredstem cells+sensors; (Matrigel™+cytokines bound to matrigel)+normalvascular stem cells+engineered support cells+sensors; and(Matrigel™+cytokines bound to Matrigel™)+normal vascular stemcells+engineered stem cells+sensors.

One embodiment of the invention includes an ATS having stem cells as aconstituent. Stem cells, which are the basic cellular building blocks oftissues and therefore organisms, are currently being considered forreplacement of damaged or non-functional cells and tissues in variousdiseases. These stem cells are extremely plastics cells, and can beinduced by a variety of proteins (cytokines and growth factors), drugsand matrices to proliferate and differentiate into specific adult cells.These stem cells can be included in the ATS for use in conjunction withimplantable devices. For example, various combinations of stem cellsand/or engineered stem cells, in association with other categories ofcells, factors and matrices, may be used to create an extremelyversatile ATS for implantable devices. These stem cells will be used todevelop a device friendly environment by 1) insitu proliferation anddifferentiation into the ATS, 2) inducing specific in growth devicefriendly cells, matrices and factors from the tissue surrounding theimplanted ATS-device and 3) inhibiting the in growth of cells, matricesand factors from the tissue surrounding the implanted AST-device, and 4)functioning as a support cell system to nurture and replace non-stemcell populations in the ATS.

One embodiment of the present invention includes the prophetic examplesof how stem cells could be used in ATS to enhance the function and lifespan of an implantable device placed into the ATS. For example, vascularendothelial cell (VEC) stem cells could be used to induceneovascularization in ATS to thereby enhance the function of implantablesensors. Experimentally, as shown in FIG. 40, this can be accomplishedby obtaining surgically the long bones from adult mice. The ends of thebones are then removed. Next, tissue culture media is perfused thru thebones to flush out bone marrow cells, which are the source of thevascular endothelial cells stem cells. The bone marrow cells arecultured in vitro for several days in the presences of the cytokines,VEGF and GM-CSF, to induce differentiation of the bone marrow cells intovascular endothelial cells. The induction of the endothelial cellphenotype is detected using Immunoassays (immunocytochemistry or FACSanalysis) to detect expression of CD34 on the surface of the vascularendothelial cells. The CD34 positive cells (CD34+) are then incorporatedinto a matrix material such as, Matrigel™, and assembled with theimplantable glucose sensor as described above. The resulting ATS(withstem cells)-sensor complex is then implanted SQ into the neck of therecipient mice.

In one embodiment of the present invention, the ATS-sensor complex canbe assembled in vivo but first injecting the Matrigel™-stem cellcombination into the skin of the mice followed by the insertion of thesensor into the matrigel-cell mixture preinjected into the mouse skin.

In one embodiment of the present invention, at various days postimplantation, for example at 1, 3, 7, 14, 30, 60, and 180 days, the miceare evaluated for glucose sensor function as previously described in theexperimental examples. For example, for sensor function evaluation, themice are injected i.p. with a glucose solution at pre-selected times theblood glucose levels as determined directly and correlated with theoutput of the implanted sensors. As controls for these studies sensorcan be implanted without any stem cell ATS, and with only Matrigel™ andthe device.

One embodiment of the invention includes using human blood mononuclearcells as a source of stem cells. Experimentally these human bloodmonocytes are isolated using Histopaque and cultured in basicendothelial cell media (Clonetics) with EGF, VEGF, FGF-b IGF-1, ascorbicacid and heparin for 8-12 week to induce endothelial stem cells. Theresulting endothelial cells will be verified using anti-CD34immunoassays, and combined with a matrix material, for exampleMatrigel™, to form the basic ATS system. The vascular endothelial cellstem cell-Matrigel™ ARS can be assembled with the glucose sensor invitro or in vivo as described in the above experimental examples. The invivo function of the ATS-Sensor complex can be tested in nude mice. Nudemice (athymic) will be used to prevent rejection of the human stem cellssince the recipient host is murine not human. Once these experimentalstudies are completed studies using human stem cells based ATS-sensorassemblies can be done, for example, in human hosts.

One embodiment of the present invention includes using an ATS havingvascular endothelial cell (VEC) stem cells and a matrix material, forexample Matrigel™ in addition to including cytokines and/or cytokineproducing support cells, to Induce, for example neovascularization forthe purpose of enhancing the function of implantable sensors.

One embodiment of the invention includes using the ability of the localexpression of SDF-1, a know inducer of endothelial stem cells, to induceneovascularization in conjunction with vascular endothelial stem cellsin vivo. For these studies two general approaches may be used.

In the first approach, an addition of recombinant SDF-1 to, for example,Matrigel™ is made prior to the addition of the stem cells and glucosesensor, to create a depot of SDF-1 which will stimulate thedifferentiation of the stem cells into vascular endothelial cells thatwill directly promote neovascularization and or directly form bloodvessel (vasculogenesis). Implantation into, for example, mice can the bemade and the mice can be evaluated at various times for glucose sensorfunction as described above.

In a second approach mouse fibroblasts which over-express SDF-1 can beprepared in a fashion similar as was done for the VEGF over-expressingfibroblast as previously described. The SDF-1 over expressing fibroblastcan then be mixed with vascular endothelia cell stems cells in a matrixmaterial, for example Matrigel™ in order to form the ATS. The ATS canthen be combined with the glucose sensor in vitro or in vivo and thenimplanted into the mice and evaluated at various times for glucosesensor function as described previously.

On embodiment of the present invention includes an ATS havinggenetically engineered vascular endothelial cell (VEC) stem cells and amatrix material to induce neovascularization thereby enhancing thefunction of implantable sensors

One embodiment of the present invention includes using stem cells toinduce neovascularization in vivo at sites of sensor implantation byengineering the stem cells themselves to over express angiogenic factorsand/or stem cell factors. For this approach stem cell models asdescribed above can be used. However, the cells can be geneticallyengineered such that the stem cells over produce pro-neovascularizationcytokines such as VEGF and/or stem cell factors such as SDF-1.

One embodiment of the present invention includes combining various stemcells and engineered stem cells in the a matrix material, for exampleMatrigel™, to form and ATS in order to enhance neovascularization andsensor function in vivo. For example, the following combinations couldbe made:

-   -   Combination 1: Normal VEC stem cells plus VEGF-over expressing        stem cells.    -   Combination 2: normal VEC stem cells plus SDF-1 over expressing        Stem cells    -   Combination 3: normal VEC stem cells plus VEGF-over expressing        stem cells and SDF-1 over expressing Stem Cells        The various combination of normal and engineered stem cells        described above can be added to the Matrigel™ to form an ATS        which is then combined with the glucose sensor in vitro or in        vivo. An implantation is made into mice and evaluated at various        times for glucose sensor function as described above.

In one embodiment of the invention there are a wide variety of methodsemployed for gene transfer in cells and tissue. Three of the majorapproaches used include: 1) plasmid based gene transfer, 2) retrovectorbased gene transfer and adenovector based gene transfer. Plasmid basedgene transfer utilizes “naked” DNA to directly transfer geneticinformation into cells in vitro and or in vivo. Plasmid based genetransfer has the advantage that it is simple, but it is extremelyinefficient particularly in vivo. Retrovector based gene transferutilizes retroviral vectors to “carry” the selected genetic informationinto the cells via specific virus receptors on the surface of targetcells. Retrovectors have the advantage in that they are extremelystable, but they require a selection procedure, which identifies cellsin which the genes have successfully been transferred. Adenovectors,like retrovectors utilizes adenoviral vectors to “carry” the selectedgenetic information into the cells via specific virus receptors on thesurface of target cells. Adenovectors have the advantage of being veryefficient in gene transfer also the gene expression may be transient.Generally for gene therapy, adenovectors have been the system of choice.Adenovirus binds to a surface receptor known as CAR, and CARs have beenidentified on human (hCAR) and murine cells. Unfortunately not all cellshave high enough levels of CAR to allow infection with adenovectors,thus limiting the spectrum of target cells in which gene transfer can beachieved. For example, generally fibroblasts have limited levels of CARand thus are not used as target cells for adenovector based genetransfer. For the ATS a protocol for genes transferred into target cellsthat are CAR deficient, thus allowing the use of adenovector in thesecells.

For example, as shown in FIG. 42, a prophetic experiment includes anadenovector based gene transfer in cells that contain little or no CARcan be achieved by first transferring the CAR gene into the CARdeficient cell, demonstrating CAR expression, and finally transferringthe gene of choice into the CAR transfected cell. To achieve this canfirst culture NIH 3T3 mouse fibroblasts at a concentration of 3E5 cells.Supercoiled plasmid containing mouse CAR or human CAR genes is thenadded to the cells in DMEM containing lipofectamine and incubated atroom temperature. Next, the cells-plasmid-lipofectamine culture istransferred to 37° C. and 5% CO₂ for 5 hours. After the 5 hr time frame,2 ml of 10% FBS is added to the culture and it is incubated at 37° C.and 5% CO₂. CAR expression in the cells is then determined byimmunocytochemistry and western blot technology using an antibody thatis specific for mouseCAR (mCAR). The resulting mCAR positive cells canbe tested for gene transfer using adenoviral vectors containing greenfluorescence protein gene (GFP), human VEGF genes or mouse VEGF genes byincubation of the CAR positive 3T3 fibroblast with these viral vectorsindividually. The resulting transfected cells can be tested forsuccessful gene transfer by 1) evaluating the GFP transfected cells forgreen fluorescence appearance under direct microscopic evaluation; 2)evaluating expression of mouse VEGF expression by immunoassays (ELISAand Western blot) using an antibody specific for mouse VEGF; 3)evaluating expression of human VEGF expression by immunoassays (ELISAand Western blot) using an antibody specific for human VEGF. Theresulting mouse VEGF or human VEGF 3T3 fibroblast can then be added tomatrigel and inject s.q. in the skin of mice to determine there abilityto induce new blood vessel formation using standard histologicalevaluation of the tissue obtained from the site of implantation. The 3T3cells that successfully induce new blood vessel formation in the mouseskin can then be used in the ATS with the implantable glucose sensor todetermine the ability of these genetically engineered cells to enhanceglucose sensor function in vivo.

In one embodiment of the present invention artificial skin may beutilized. Artificial skin, as a tool to promote the regeneration andrepair of injured tissues, e.g. skin of burn victims, and repair ofulcerated skin, has seen growing acceptance in the medical community inrecent years. Initially artificial skins were composed of temporaryacellular materials, which where used to protect the injured skin frominfection and dehydration, while repair and regeneration of the skinwere occurring. More recently artificial skins have become increasingcomplex assemblies of synthetic polymers and natural products, includingcells. These “artificial skins” can utilized to enhance and or protectimplantable devices, such as glucose sensors by including the use ofvarious techniques, teachings, suggestions, and protocols disclosedherein.

While preferred embodiments of the foregoing invention have been setforth for purposes of illustration, the foregoing description should notbe deemed a limitation of the invention herein. Accordingly, variousmodifications, adaptations and alternatives may occur to one skilled inthe art without departing from the spirit and scope of the presentinvention.

What is claimed is:
 1. A method comprising: obtaining an implantabledevice comprising a sensor having an implantable portion, forming animplantation pocket at an implantation site in a biological tissue bysubcutaneously injecting a liquid into subcutaneous biological tissue,making an incision in the implantation pocket, dipping the implantableportion of the sensor in a basement membrane, inserting the dippedportion of the sensor at the location of the incision into theimplantation pocket in the subcutaneous biological tissue, andperiodically sensing changes in a level of an analyte in thesubcutaneous biological tissue using the sensor.
 2. The method of claim1, wherein the sensor is a glucose sensor.
 3. The method of claim 1,wherein the sensor is an acetaminophen sensor.
 4. The method of claim 1,wherein the basement membrane is a liquid.
 5. The method of claim 1,wherein the basement membrane is a gel.
 6. The method of claim 1,wherein the basement membrane is cell culture derived.
 7. The method ofclaim 1, wherein the sensor includes a support portion, and the methodfurther comprises securing the support portion to the biological tissue.8. The method of claim 1, wherein the sensor is configured to be fullyembedded in the biological tissue.
 9. The method of claim 1, wherein theliquid comprises a saline solution.
 10. A method of implanting a devicein a biological tissue, comprising: obtaining an implantable devicecomprising a sensor having an implantable portion and a support portion,forming an implantation pocket at an implantation site in a biologicaltissue by subcutaneously injecting an aqueous solution into subcutaneousbiological tissue, making an incision in the implantation pocket,dipping the implantable portion of the sensor in a solubilized cellculture derived basement membrane, inserting the dipped portion of thesensor at the location of the incision into the implantation pocket inthe subcutaneous biological tissue, securing the support portion of thesensor to the biological tissue, and periodically sensing changes in alevel of an analyte in the subcutaneous biological tissue using thesensor.
 11. The method of claim 10, wherein the solubilized cell culturederived basement membrane comprises collagen and laminin.
 12. The methodof claim 10, wherein the sensor is a glucose sensor.
 13. The method ofclaim 10, wherein the basement membrane is a liquid.
 14. The method ofclaim 10, wherein the basement membrane is a gel.
 15. A methodcomprising: obtaining an implantable device comprising a sensor havingan implantable portion, forming an implantation pocket at animplantation site in a biological tissue by subcutaneously injecting aliquid into subcutaneous biological tissue, making an incision in theimplantation pocket, inserting solubilized basement membrane into theimplantation pocket, and inserting the implantable portion of the sensorinto the implantation pocket at the location of the incision, andperiodically sensing changes in a level of an analyte in thesubcutaneous biological tissue using the sensor.
 16. The method of claim15, wherein the sensor is a glucose sensor.
 17. The method of claim 15,wherein the basement membrane is a liquid.
 18. The method of claim 15,wherein the sensor includes a support portion, and the method furthercomprises securing the support portion to the biological tissue.
 19. Themethod of claim 15, wherein the sensor is configured to be fullyembedded in the biological tissue.
 20. The method of claim 15, whereinthe solubilized cell culture derived basement membrane comprisescollagen and laminin.